Secreted alkaline phosphatase: an internal ... - Semantic Scholar
Secreted
alkaline
standard
phosphatase:
for expression
in the Xenopus SURESH
S. TATE,1
SIDNEY
Xenopus
REIKO
of Molecular
oocyte
is widely
of eukaryotic
being
also cDNAs
URADE,
Biology,
cell
used
RADMILA
time
used increasingly
encoding is that from
amount of
the
proteins
and
same
In
cloning
there
are
taken
vary this
various
no
at
It is of
struc-
of the Xenopus
oocytes frog
synthesized
mRNA.
the
function.
in expression for which
individual
of protein
injected
oocyte
the
same
considerably
in
the
the same amount
from report
we
describe
the
preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily
determined
using
in
a standard
the
medium
colorimetric
of cultured assay.
The
oocytes
by
amounts
of
alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes. -Tate, S. S.; Urade, R.; Micanovic, R.; Gerber, L.; Udenfriend, S. Secreted
alkaline
phosphatase:
expression of injected FASEBJ 4: 227-231; Key
Words:
MICANOVIC,
Roche Research Center,
to study
structure
tural data. One of the drawbacks system
mRNAs
oocyte
ABSTRACT
aspects
of injected LOUISE
GERBER,
AND
UDENFRIEND2
Roche Institute
The
an internal
Xenopus
for expression of injected
mRNAs 1990.
an
internal
marker
in the Xenopus
oocytes alkaline phosphatose. mRNAs
for
oocyte.
marker
The Xenopus oocyte is widely used to study numerous aspects of eukaryotic cell structure and function (1-4). The oocytes have been shown to efficiently and faithfully translate injected mRNAs. An important attribute of the Xenopus oocyte system is that it permits cloning of cDNAs encoding proteins for which no structural data are available. Many proteins have been cloned in this
0892-6638/90/0004-0227/$01
.50. © FASEB
Nutley,
New Jersey
07110,
USA
manner including receptors, ion channels, and transporters (5-9). One of the drawbacks of the Xenopus system is that individual oocytes taken at the same time from the same animal vary considerably in the amount of protein synthesized from the same amount of injected mRNA (1). There are also variations in expression among oocytes taken from different animals. Standard mRNAs to correct for variation in translation in cell-free systems are now available. To our knowledge no such internal standard has been introduced to monitor the efficiency of translation in intact cells. In an earlier report from this laboratory, the cDNA of a secreted mutant form of alkaline phosphatase (AP)3 was successfully used as a reporter gene to monitor the efficiency of transcription in cells (10). In this report we describe the preparation and use of the corresponding mRNA as an internal standard for translation of injected mRNAs in oocytes. Secreted form of human placental alkaline phosphatase (SEAP)/mRNA encodes a secreted form of human placental AP that can be readily and accurately quantified in the media of cultured oocytes by using a standard colorimetric assay. SEAP/ mRNA is, therefore, an excellent indicator of expression in oocytes when coinjected with other mRNAs. MATERIALS
AND
METHODS
All chemicals were reagent grade. L-[4,5-3H]Leucine (0.1 mCi/mol) was purchased from Amersham (Arlington Heights, Ill.). p-Nitrophenylphosphate and L-’y-glutamyl-p-nitroanilide were obtained from Sigma (St. Louis, Mo.). The 96-well flat-bottom culture plates were obtained from Corning (Corning, N.Y.). Prior to their use for oocyte culture, the wells of the plates were filled with phosphate-buffered saline (PBS) containing
‘Permanent Address: Department of Biochemistry, Cornell University Medical College, 1300 York Ave., New York, New York 10021, USA. 2To whom correspondence should be sent at the above address. 3Abbreviations: AP, alkaline phosphatase; PLAP, human placental alkaline phosphatase; SEAP, secreted form of human placental alkaline phosphatase; -y-GT, y-glutamyl transpeptidase; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
227
0.1% bovine serum albumin (BSA). After 30 mm at room temperature, the solution was removed and the wells were washed three times with PBS. This procedure minimizes losses of SEAP on the surface of the wells. Production
of SEAP/mRNA
Earlier we described a vector, pBC12/PLAP, which contains cDNA encoding the entire structure of the 513-amino acid, membrane-anchored form of human placental alkaline phosphatase (PLAP) (11). The secreted form of PLAP was produced from the full-length clone by inserting a translation-terminating codon after amino acid 489 by oligodeoxynucleotide-directed mutagenesis (10). The pBC12/PLAP489 vector thus produced was digested with EcoRI and Kpnl restriction endonucleases. The 2-kb insert encoding SEAP was isolated and subcloned into the corresponding cloning sites of the transcription vector, pGEM-4Z (Promega, Madison, Wisc.), downstream of the SP6 transcriptional promoter. The plasmid pGEM-4Z/PLAP489 was amplified, purified, and linearized by digestion with Hindlil. The linearized plasmid was transcribed, using a transcription kit purchased from Promega, in the presence of the cap structure analog, m7-GpppG. Purified SEAP/mRNA was dissolved in water to a concentration of 0.2 tg/l and stored at 70#{176}C. Usually about 2 eg of SEAP/mRNA was obtained per microgram of the vector DNA. Further dilutions of the RNA were made with Escherichia coli tRNA (1 tg/tl). In vitro translation of SEAP/mRNA in the rabbit reticulocyte lysate system in the presence of 35S-labeled methionine yielded a product exhibiting, as expected, an apparent Mr of about 57,000 on NaDodSO4/PAGE. -
Preparation
of rat kidney
mRNA
The mRNA for expression of both the leucine transporter and -y-glutamyl transpeptidase was obtained from rat kidney. Total RNA was isolated by the method of Chomczynski and Sacchi (12) from kidneys that were freshly removed from Wistar rats weighing about 200 g each. The extraction medium, RNAzo1, was purchased from CinnalBiotecx Laboratories, Friendswood, Tex. Poly(A)RNA was purified by chromatography on oligo(dT)cellulose (13) and precipitated with ethanol (80% final) in the presence of 0.3 M sodium acetate (pH 5.2). The RNA was dissolved in water, adjusted to a concentration of I sg/tl, and stored at 70#{176}C until use. -
Microinjection
of mRNA
into
oocytes
Xenopus laevis females (Nasco, Fort Atkinson, Wisc.) were anesthetized with tricaine (3-aminobenzoic acid ethyl ester), and ovarian follicles were surgically removed using standard procedures (2). Follicle cells were dispersed and individual oocytes were released by incubation at 20#{176}C for 2 h in a Ca2-free oocyte medium
228
Vol. 4
Feb. 1990
(96 mM NaCl, 2 mM KC1, 1 mM MgC12, 5 mM Hepes, pH 7.6) (14) containing collagenase (2 mg/mI; Sigma, type 1A). The oocytes were extensively washed and allowed to recover overnight at 20#{176}C in culture dishes in Ca2-supplemented (1.8 mM) oocyte medium containing 2.5 mM sodium pyruvate, penicillin (100 units/ml), and streptomycin (100 jzg/ml). Oocytes (stages V-VI) were selected and microinjected with either 50 nl of water (controls) or 50 ni of solution containing either SEAP/mRNA, rat kidney mRNA, or a mixture of the two mRNAs (approximately 5-50 ng of each mRNA). The injected oocytes were maintained at 20#{176}C in the same Ca2-containing medium for about 18 h, at which time damaged oocytes were discarded. The remaining oocytes were individually transferred to wells of 96-well flat-bottom culture plate and maintained in 0.2 ml of the oocyte medium at 20#{176}C. During the time of incubation, the medium from each well was removed for AP assay every 24 h and replaced by an equal amount of fresh oocyte medium. Alkaline
phosphatase
assay
Levels of AP in the oocyte culture medium were assayed by monitoring the rate of hydrolysis of pnitrophenylphosphate essentially as described by Berger et al. (10). An aliquot (100 1d) of the medium from each oocyte was transferred to a well of a 96-well plate containing 100 l of buffer (1 M diethanolamine, pH 9.8, 0.5 mM MgCl2, 0.02 mM ZnSO4, and 20 mM Lhomoarginine (to inhibit the small amounts of nonspecific AP normally present in the oocytes). When all the samples had been added, the culture plate was warmed to 37#{176}C for 10 mm. Forty microliters of warmed 60 mM p-nitrophenylphosphate was added to each well with mixing and the plate was incubated at 37#{176}C. Absorbance at 405 nm was recorded for every well every 10 mm in an Artek automatic plate reader to determine the linear reaction rate. One milliunit of SEAP is the amount of enzyme that catalyzes the formation of 1 nmol of p-nitrophenol per hour. Once the relationship between the expression of a given mRNA and SEAP/mRNA is established, it is no longer necessary to measure absorbance; visual examination of the yellow product (p-nitrophenol) produced in assay mixtures in the culture plates is sufficient to permit selection of those oocytes that express mRNA efficiently.
Uptake
of L-leucine
by oocytes
The System L amino acid transporter is one of several membrane-associated mechanisms for cellular uptake of amino acids (15). The System L transporter is generally assayed by monitoring the uptake of radioactive amino acids such as [3H]leucine. The Na-independent uptake of L-leucine by oocytes was determined as described (16). The oocytes (up to 8) injected with either water (control), SEAP/mRNA, or rat kidney/ mRNA were rinsed thoroughly with Na-free oocyte
The FASEB Journal
TATE ET AL.
medium (96 mM choline chloride, 2 mM KC1, 1 mM MgCl2, 1.8 mM CaC12, 5 mM Hepes, pH 7.6) and then transferred to 1 ml of the Na-free medium containing 0.2 mM L-[4,5-3H}leucine (final specific activity in transport medium, 100 tCi/tmol). After incubation for 10 mm at 20#{176}C, uptake was terminated by removing the incubation medium and washing the oocytes with 4 x 4-mi aliquots of ice-cold Na-free medium. Each oocyte was dissolved in 0.5 ml of 2% NaDodSO4, and was assayed by scintillation spectrometry. Uptake is expressed as pmol of L-ieucine per oocyte per 10 mm. For oocytes injected with a mixture of SEAP and rat kidney mRNAs, SEAP activity in the culture medium from each oocyte was first assayed as described. Individual oocytes were then separately rinsed with the Na-free oocyte medium and each oocyte incubated for 10 mm at 20#{176}C in 0.2 ml of the Na-free medium con-
A
B f
taming 0.2 mM L-[3H]leucine. The oocytes were then individually washed and uptake determined as described above. -y-Glutamyl
transpeptidase
activity
The activity of this cell-surface enzyme that catalyzes the first step in the degradation of glutathione was determined essentially as described (17). Four oocytes from each group were placed in 0.4 ml of ‘y-GT assay solution (0.05 M Tris-HC1, pH 8.0, 0.05 M NaC1, 10 mM glycylglycine, and 0.5 mM L-7-giutamyi-p-nitroanilide) prewarmed to 37#{176}C. After incubation at 37#{176}C for 3 h, a 0.2-mi aliquot of the assay mixture was removed, mixed with 0.2 ml of water, and absorbance at 405 nm measured in a Bausch and Lomb Spectronic 88 spectrophotometer. y-Glutamyl transpeptidase activity is expressed as the average of such determinations on eight oocytes from each group of microinjected oocytes. One milliunit of -y-GT is the amount of enzyme that catalyzes the release of 1 nmol of p-nitroaniline per hour.
120
RESULTS 80
10
:1
‘U
C
60
40
20
LI r
Figure 1. Variation in expression among oocytes injected at the same time with the same amount of a given mRNA. A and B) Secretion of SEAP by oocytes, each of which was injected with either 5 ng or 10 ng of SEAP/mRNA. The two groups of oocytes were taken from individual frogs. C) Expression of L-leucine uptake by oocytes each injected with 33 ng of rat kidney mRNA. The uptake of L-leucine by control (water-injected) (6 pmol per oocyte) oocytes has been subtracted. Measurements were made on individual oocytes 48 h after injection of the mRNAs. Each point represents one oocyte.
SECRETED ALKALINE
PHOSPHATASE
In these studies, the first measurements of the proteins expressed from exogenous mRNAs were generally made 20-24 h after injection. At this time, appreciable SEAP was already in the medium.4 SEAP continued to accumulate in the medium during the entire period of the experiment, generally up to 72 h after injection. SEAP secreted by oocytes injected with the same amount of SEAP/mRNA varied considerably among oocytes isolated from the same Xenopus. Thus, in one experiment in which 5 ng of SEAP/mRNA was injected per oocyte, SEAP in the medium after 48 h varied from as little as 5 milliunits to as much as 62 milliunits (average about 34 milliunits SEAP/oocyte) (Fig. 1A). In a similar experiment in which 10 ng of SEAP/mRNA was injected per oocyte, the values of SEAP ranged from about 8 to 150 milliunits (average about 85 milliunits) (Fig. 1B). In both experiments, about 20% of the oocytes failed to secrete detectable amounts of SEAP. Similarly, in oocytes injected with rat kidney mRNA, the levels of expression of Na-independent L-leucine uptake varied over a wide range. Thus, in oocytes injected with 33 ng of the kidney mRNA, the uptake of L-leucine after 48 h varied from about 5 pmol to as much as 65 pmol of L-leucine/(oocyte. 10 mm) (Fig. 1C). Again, about one-fifth of the injected oocytes failed to express Lleucine uptake significantly above that observed in controls (H20 injected) oocytes. No SEAP activity was de-
4When monitoring the expression of proteins with a high turnover rate, it might be desirable to assay SEAP in the medium at earlier times after the injection of mRNAs. In such situations, depending on the amount of SEAP/mRNA injected, either the colonmetric assay or a highly sensitive, bioluminescence-based assay (11) for AP can be used. Indeed, monitoring SEAP in the medium should also facilitate the kinetics of expression of secretory and membrane proteins.
229
tected in the media of either the control or the kidney mRNA-injected oocytes. Also, the oocytes injected with SEAP/mRNA alone exhibited L-leucine uptake similar to that seen in control oocytes, about 5-7 pmol of Lieucine/(oocyte. 10 mm). When SEAP and rat kidney mRNA (3 and 33 ng, respectively) were coinjected into oocytes (Fig. 2), a good correlation was observed between the amount of SEAP and transport of L-leucine (correlation coefficient 0.84). Thus, oocytes that secreted relatively high levels of SEAP also exhibited high expression of L-leucine uptake and vice versa. A similar range of L-ieucine uptake and of SEAP was seen among oocytes injected with either rat kidney mRNA or SEAP/mRNA alone (Fig. 1), indicating that at the amounts used in Fig. 2, when SEAP and kidney mRNAs are coinjected, they do not influence each other’s expression. Figure 2 also shows that expression of -y-GT correlates well with SEAP in oocytes coinjected with SEAP and kidney mRNA. In control oocytes and in oocytes injected with SEAP/ mRNA alone, no y-GT was detected. In a separate experiment, each oocyte in the group was coinjected with 1.5 ng of SEAP/mRNA and 25 ng of kidney mRNA. Here again a good correlation was observed between SEAP and L-leucine uptake (correlation coefficient = 0.93) and between SEAP and -y-GT. =
I
I
I
I
I
I
60-
2.4
2.0
50-
. ,
40-
1.6
.
:
30-
.
1.2
#{149}
fa”
0.8
#{149}..-; 10
.
#{149}
1)
15
2)
25
3
35
40
SEAP in medium (munits) Figure 2. Correlation between SEAP activity in the medium and L-leucine uptake by individual oocytes coinjected with a mixture of SEAP/mRNA and rat kidney mRNA (3 ng and 33 ng, respectively, per oocyte) (filled circles). Measurements were made 72 h after injection. The open circle denotes the average value for L-leucine uptake by control (water-injected) oocytes. Bars show the average glutamyl transpeptidase (‘y-GT) activity in oocytes coinjected with kidney and SEAP mRNA. The horizontal reach of the bar denotes the range of SEAP secreted by individual oocytes. For y-GT, four oocytes expressing similar levels of SEAP were pooled and assayed as described in Materials and Methods. Each bar represents the average of two such determinations (eight oocytes). The shaded area highlights the correlation between SEAP and the expression of L-leucine transport.
Vol. 4
DISCUSSION The relative stability, high substrate turnover number, and high affinity for a range of substrates have made AP a widely used reagent in many diagnostic and biochemical procedures. These properties help make AP a useful indicator of expression in the Xenopus oocyte system (and, presumably, in other expression systems). Since, like most other cells, oocytes have significant amounts of cell-surface AP (unpublished data), use of the wild-type (membrane-bound) enzyme has limited usefulness as an indicator of expression of foreign mRNAs. To overcome this problem, we used the mRNA encoding a secreted mutant form of human placental alkaline phosphatase. The cDNA encoding SEAP was cloned into a vector that allows large-scale synthesis of SEAP/mRNA in vitro.5 This mutant form of AP is secreted by the oocytes into the medium, thus allowing rapid identification of oocytes that efficiently express the injected mRNAs. An advantage of using the placental form of AP is that, in contrast to other isozymes (including the oocyte enzyme), it is unaffected by 10 mM homoarginine (11). Thus, inclusion of homoarginine in assay media eliminates the possibility of interference by endogenous forms of AP. Injection of as little as 1 ng of SEAP/mRNA per oocyte resulted in the secretion of readily measurable amounts of SEAP into the medium. Using Na-independent L-leucine transport and y-GT (both expressed in oocytes from rat kidney mRNA), we have shown that expression of these two membrane-associated functions and secretion of SEAP parallel each other in oocytes injected with the two mRNAs. The small amounts of SEAP/mRNA used do not interfere with the expression of the test mRNAs SEAP is secreted
-
5
230
In this experiment, SEAP in the medium from individual oocytes varied from about 2 to 27 milliunits, and L-leucine uptake for the corresponding oocytes ranged from 8 to 70 pmol/(oocyte. 10 mm); y-GT activity varied from about 0.2 milliunits per oocyte for the low SEAP expressors to about 2 milliunits per oocyte for the high expressors of SEAP.
Feb. 1990
and the vice medium versa. Furthermore, into and easily
since quan-
tified, each oocyte is available for assay of proteins expressed either on the oocyte surface or intracellularly. SEAP in the medium can be assayed by using standard colorimetric procedures and a number of readily available scanning instruments. For most purposes, however, visual examination of the assay mixtures allows rapid selection of those oocytes that efficiently express the injected mRNAs. Indeed, in our investigations of the cloning and characterization of the mammalian amino acid transporters, we now routinely coinject SEAP/mRNA with the test mRNA (either cellular or that synthesized in vitro from cDNA libraries) and use a visual test for SEAP activity in the medium to quickly
5The vector, pGEM-4Z/PLAP489, be provided to investigators upon
The FASEB Journal
containing SEAP/cDNA request to S. U.
will
TATE ET AL.
select oocytes for amino acid uptake assay (usually those expressing SEAP above the median). The inherent difficulties usually encountered in the use of the oocyte system, viz, the large variations in expression of foreign mRNAs by individual oocytes from the same animal and among batches from different animals, are thus overcome. The reduction in the number of oocytes that need to be assayed results in an enormous saving of time and expense. Since injection of too much mRNA or poor quality mRNA suppresses translation of both endogenous and exogenous mRNAs, diminished expression of SEAP/mRNA under these conditions signals the investigator to potential problems. SEAP/mRNA will be particularly valuable as a predictive internal indicator of the expression of exogenous mRNAs in oocytes in investigations that involve complex measurements (e.g., electrophysiological, binding assays, etc.) on individual or pooled oocytes. In such experiments, the investigator should select only those oocytes that express the highest amounts of SEAP (e.g., upper 10-20%). SEAP/mRNA will also be useful in other expression systems such as those that use sea urchin eggs and newt (Cynops) oocytes. Recently, a method for the introduction of RNA into tissue culture cells using cationic iiposomes (lipofectin) was introduced (18). This procedure allows RNA transfection of a wide variety of mammalian, amphibian, and insect cells and provides a new approach to the study of factors that influence the translation and stability of eukaryotic mRNAs. SEAP/mRNA may also prove to be a reliable indicator of the efficiency of this transfection process. Thus, we anticipate many uses for SEAP/ mRNA as an internal standard in studies involving expression of eukaryotic mRNAs.
3. 4. 5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16.
REFERENCES 1. Gurdon, oocytes 370-386 2. Colman, Xenopus
J. B., and Wickens, M. P. (1983) The use of Xenopus for the expression of cloned genes. Methods EnzymoL 101,
17. 18.
A. (1984) Translation oocytes. Transcription
of eukaryotic and Translation:
messenger A Practical
RNA in Approach
(Hames, B. D., and Higgins, S. J., eds) pp. 271-300, IRL Press, Washington, DC Melton, D. A. (1987) Translation of messenger RNA in injected frog oocytes. Methods Enzymol. 152, 288-296 Snutch, T. P. (1988) The use of Xenopus oocytes to probe synaptic communication. Trends Neurosci. 11, 250-256 Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., and Nakanishi, S. (1987) cDNA cloning of bovine substance K receptor through oocyte expression system. Nature (London) 329, 836-838 Hediger, M. A., Coady, M. J., Ikeda, T. S., and Wright, E. M. (1987) Expression cloning and cDNA sequencing of Na/ glucose co-transporter. Nature (London) 330, 379-381 Lubbert, H., Hoffmann, B. J., Snutch, T. P., Van Dyke, T., Levine, A. J., Hartig, P. R., Lester, H. A., and Davidson, N. (1987) cDNA cloning of a serotonin 5-HT,c receptor by dcctrophysiological assays of mRNA-injected Xenopus oocytes. Proc. NatI. Acad. Sd. USA 84, 4332-4336 Julius, D., MacDermott, A. B., Axel, R., and Jessell, T. M. (1988) Molecular characterization of a functional cDNA encoding the serotonin Ic receptor. Science 241, 558-564 Takumi, T., Ohkubo, H., and Nakanishi, 5. (1988) Cloning of a membrane protein that induces a slow voltage-gated potassium current. Science 242, 1042-1045 Bergen, J., Hauber, J., Hauber, R., Geiger, R., and Cullen, B. R. (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66, 1-10 Berger,J., Howard, A. D., Gerger, L., Cullen, B. R., and Udenfriend, 5. (1987) Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. Proc. NatI. Acad. Sd. USA 84, 4885-4889 Chomczynski, P., and Sacchi, N. (1987) Single-step method for RNA isolation by acid guanidium thiocyanate-phenol-chioroform extraction. AnaL Biochem. 162, 156-159 Badley, J. E., Bishop, G. A., St. John, T., and Frelinger, J. A. (1988) A simple, rapid method for the purification of poly A RNA. Biotechniques 6, 114-116 Wallace, R. A., Jared, D. W., Dumont, J. N., and Sega, M. W. (1973) Protein incorporation by isolated amphibian oocytes: optimum incubation conditions. j Exp. Zool. 184, 321-334 Christensen, H. N. (1985) On the strategy of kinetic discrimination of amino acid transport systems. J. Membr. BioL 84, 9 7-103 Tate, S. S., Urade, R., Getchell, T. V., and Udenfniend, S. (1989) Expression of the mammalian Na-independent L system amino acid transporter in Xenopus laevis oocytes. Arch. Biochem. Biophys. 275, 591-596 Tate, S. S., and Meister, A. (1985) ‘y-Glutamyl transpeptidase from kidney. Methods Enzymol. 113, 400-419 Malone, R. W., Felgner, P. L., and Verma, I. M. (1989) Cationic liposome-mediated RNA transfection. Proc. Natl. Acad. Sci. USA 86, 6077-6081
Receivedfor publication October 26, 1989. Acceptedfor publication November 22, 1989.
SECRETED ALKALINE
PHOSPHATASE
231
alkaline
standard
phosphatase:
for expression
in the Xenopus SURESH
S. TATE,1
SIDNEY
Xenopus
REIKO
of Molecular
oocyte
is widely
of eukaryotic
being
also cDNAs
URADE,
Biology,
cell
used
RADMILA
time
used increasingly
encoding is that from
amount of
the
proteins
and
same
In
cloning
there
are
taken
vary this
various
no
at
It is of
struc-
of the Xenopus
oocytes frog
synthesized
mRNA.
the
function.
in expression for which
individual
of protein
injected
oocyte
the
same
considerably
in
the
the same amount
from report
we
describe
the
preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily
determined
using
in
a standard
the
medium
colorimetric
of cultured assay.
The
oocytes
by
amounts
of
alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes. -Tate, S. S.; Urade, R.; Micanovic, R.; Gerber, L.; Udenfriend, S. Secreted
alkaline
phosphatase:
expression of injected FASEBJ 4: 227-231; Key
Words:
MICANOVIC,
Roche Research Center,
to study
structure
tural data. One of the drawbacks system
mRNAs
oocyte
ABSTRACT
aspects
of injected LOUISE
GERBER,
AND
UDENFRIEND2
Roche Institute
The
an internal
Xenopus
for expression of injected
mRNAs 1990.
an
internal
marker
in the Xenopus
oocytes alkaline phosphatose. mRNAs
for
oocyte.
marker
The Xenopus oocyte is widely used to study numerous aspects of eukaryotic cell structure and function (1-4). The oocytes have been shown to efficiently and faithfully translate injected mRNAs. An important attribute of the Xenopus oocyte system is that it permits cloning of cDNAs encoding proteins for which no structural data are available. Many proteins have been cloned in this
0892-6638/90/0004-0227/$01
.50. © FASEB
Nutley,
New Jersey
07110,
USA
manner including receptors, ion channels, and transporters (5-9). One of the drawbacks of the Xenopus system is that individual oocytes taken at the same time from the same animal vary considerably in the amount of protein synthesized from the same amount of injected mRNA (1). There are also variations in expression among oocytes taken from different animals. Standard mRNAs to correct for variation in translation in cell-free systems are now available. To our knowledge no such internal standard has been introduced to monitor the efficiency of translation in intact cells. In an earlier report from this laboratory, the cDNA of a secreted mutant form of alkaline phosphatase (AP)3 was successfully used as a reporter gene to monitor the efficiency of transcription in cells (10). In this report we describe the preparation and use of the corresponding mRNA as an internal standard for translation of injected mRNAs in oocytes. Secreted form of human placental alkaline phosphatase (SEAP)/mRNA encodes a secreted form of human placental AP that can be readily and accurately quantified in the media of cultured oocytes by using a standard colorimetric assay. SEAP/ mRNA is, therefore, an excellent indicator of expression in oocytes when coinjected with other mRNAs. MATERIALS
AND
METHODS
All chemicals were reagent grade. L-[4,5-3H]Leucine (0.1 mCi/mol) was purchased from Amersham (Arlington Heights, Ill.). p-Nitrophenylphosphate and L-’y-glutamyl-p-nitroanilide were obtained from Sigma (St. Louis, Mo.). The 96-well flat-bottom culture plates were obtained from Corning (Corning, N.Y.). Prior to their use for oocyte culture, the wells of the plates were filled with phosphate-buffered saline (PBS) containing
‘Permanent Address: Department of Biochemistry, Cornell University Medical College, 1300 York Ave., New York, New York 10021, USA. 2To whom correspondence should be sent at the above address. 3Abbreviations: AP, alkaline phosphatase; PLAP, human placental alkaline phosphatase; SEAP, secreted form of human placental alkaline phosphatase; -y-GT, y-glutamyl transpeptidase; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
227
0.1% bovine serum albumin (BSA). After 30 mm at room temperature, the solution was removed and the wells were washed three times with PBS. This procedure minimizes losses of SEAP on the surface of the wells. Production
of SEAP/mRNA
Earlier we described a vector, pBC12/PLAP, which contains cDNA encoding the entire structure of the 513-amino acid, membrane-anchored form of human placental alkaline phosphatase (PLAP) (11). The secreted form of PLAP was produced from the full-length clone by inserting a translation-terminating codon after amino acid 489 by oligodeoxynucleotide-directed mutagenesis (10). The pBC12/PLAP489 vector thus produced was digested with EcoRI and Kpnl restriction endonucleases. The 2-kb insert encoding SEAP was isolated and subcloned into the corresponding cloning sites of the transcription vector, pGEM-4Z (Promega, Madison, Wisc.), downstream of the SP6 transcriptional promoter. The plasmid pGEM-4Z/PLAP489 was amplified, purified, and linearized by digestion with Hindlil. The linearized plasmid was transcribed, using a transcription kit purchased from Promega, in the presence of the cap structure analog, m7-GpppG. Purified SEAP/mRNA was dissolved in water to a concentration of 0.2 tg/l and stored at 70#{176}C. Usually about 2 eg of SEAP/mRNA was obtained per microgram of the vector DNA. Further dilutions of the RNA were made with Escherichia coli tRNA (1 tg/tl). In vitro translation of SEAP/mRNA in the rabbit reticulocyte lysate system in the presence of 35S-labeled methionine yielded a product exhibiting, as expected, an apparent Mr of about 57,000 on NaDodSO4/PAGE. -
Preparation
of rat kidney
mRNA
The mRNA for expression of both the leucine transporter and -y-glutamyl transpeptidase was obtained from rat kidney. Total RNA was isolated by the method of Chomczynski and Sacchi (12) from kidneys that were freshly removed from Wistar rats weighing about 200 g each. The extraction medium, RNAzo1, was purchased from CinnalBiotecx Laboratories, Friendswood, Tex. Poly(A)RNA was purified by chromatography on oligo(dT)cellulose (13) and precipitated with ethanol (80% final) in the presence of 0.3 M sodium acetate (pH 5.2). The RNA was dissolved in water, adjusted to a concentration of I sg/tl, and stored at 70#{176}C until use. -
Microinjection
of mRNA
into
oocytes
Xenopus laevis females (Nasco, Fort Atkinson, Wisc.) were anesthetized with tricaine (3-aminobenzoic acid ethyl ester), and ovarian follicles were surgically removed using standard procedures (2). Follicle cells were dispersed and individual oocytes were released by incubation at 20#{176}C for 2 h in a Ca2-free oocyte medium
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(96 mM NaCl, 2 mM KC1, 1 mM MgC12, 5 mM Hepes, pH 7.6) (14) containing collagenase (2 mg/mI; Sigma, type 1A). The oocytes were extensively washed and allowed to recover overnight at 20#{176}C in culture dishes in Ca2-supplemented (1.8 mM) oocyte medium containing 2.5 mM sodium pyruvate, penicillin (100 units/ml), and streptomycin (100 jzg/ml). Oocytes (stages V-VI) were selected and microinjected with either 50 nl of water (controls) or 50 ni of solution containing either SEAP/mRNA, rat kidney mRNA, or a mixture of the two mRNAs (approximately 5-50 ng of each mRNA). The injected oocytes were maintained at 20#{176}C in the same Ca2-containing medium for about 18 h, at which time damaged oocytes were discarded. The remaining oocytes were individually transferred to wells of 96-well flat-bottom culture plate and maintained in 0.2 ml of the oocyte medium at 20#{176}C. During the time of incubation, the medium from each well was removed for AP assay every 24 h and replaced by an equal amount of fresh oocyte medium. Alkaline
phosphatase
assay
Levels of AP in the oocyte culture medium were assayed by monitoring the rate of hydrolysis of pnitrophenylphosphate essentially as described by Berger et al. (10). An aliquot (100 1d) of the medium from each oocyte was transferred to a well of a 96-well plate containing 100 l of buffer (1 M diethanolamine, pH 9.8, 0.5 mM MgCl2, 0.02 mM ZnSO4, and 20 mM Lhomoarginine (to inhibit the small amounts of nonspecific AP normally present in the oocytes). When all the samples had been added, the culture plate was warmed to 37#{176}C for 10 mm. Forty microliters of warmed 60 mM p-nitrophenylphosphate was added to each well with mixing and the plate was incubated at 37#{176}C. Absorbance at 405 nm was recorded for every well every 10 mm in an Artek automatic plate reader to determine the linear reaction rate. One milliunit of SEAP is the amount of enzyme that catalyzes the formation of 1 nmol of p-nitrophenol per hour. Once the relationship between the expression of a given mRNA and SEAP/mRNA is established, it is no longer necessary to measure absorbance; visual examination of the yellow product (p-nitrophenol) produced in assay mixtures in the culture plates is sufficient to permit selection of those oocytes that express mRNA efficiently.
Uptake
of L-leucine
by oocytes
The System L amino acid transporter is one of several membrane-associated mechanisms for cellular uptake of amino acids (15). The System L transporter is generally assayed by monitoring the uptake of radioactive amino acids such as [3H]leucine. The Na-independent uptake of L-leucine by oocytes was determined as described (16). The oocytes (up to 8) injected with either water (control), SEAP/mRNA, or rat kidney/ mRNA were rinsed thoroughly with Na-free oocyte
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TATE ET AL.
medium (96 mM choline chloride, 2 mM KC1, 1 mM MgCl2, 1.8 mM CaC12, 5 mM Hepes, pH 7.6) and then transferred to 1 ml of the Na-free medium containing 0.2 mM L-[4,5-3H}leucine (final specific activity in transport medium, 100 tCi/tmol). After incubation for 10 mm at 20#{176}C, uptake was terminated by removing the incubation medium and washing the oocytes with 4 x 4-mi aliquots of ice-cold Na-free medium. Each oocyte was dissolved in 0.5 ml of 2% NaDodSO4, and was assayed by scintillation spectrometry. Uptake is expressed as pmol of L-ieucine per oocyte per 10 mm. For oocytes injected with a mixture of SEAP and rat kidney mRNAs, SEAP activity in the culture medium from each oocyte was first assayed as described. Individual oocytes were then separately rinsed with the Na-free oocyte medium and each oocyte incubated for 10 mm at 20#{176}C in 0.2 ml of the Na-free medium con-
A
B f
taming 0.2 mM L-[3H]leucine. The oocytes were then individually washed and uptake determined as described above. -y-Glutamyl
transpeptidase
activity
The activity of this cell-surface enzyme that catalyzes the first step in the degradation of glutathione was determined essentially as described (17). Four oocytes from each group were placed in 0.4 ml of ‘y-GT assay solution (0.05 M Tris-HC1, pH 8.0, 0.05 M NaC1, 10 mM glycylglycine, and 0.5 mM L-7-giutamyi-p-nitroanilide) prewarmed to 37#{176}C. After incubation at 37#{176}C for 3 h, a 0.2-mi aliquot of the assay mixture was removed, mixed with 0.2 ml of water, and absorbance at 405 nm measured in a Bausch and Lomb Spectronic 88 spectrophotometer. y-Glutamyl transpeptidase activity is expressed as the average of such determinations on eight oocytes from each group of microinjected oocytes. One milliunit of -y-GT is the amount of enzyme that catalyzes the release of 1 nmol of p-nitroaniline per hour.
120
RESULTS 80
10
:1
‘U
C
60
40
20
LI r
Figure 1. Variation in expression among oocytes injected at the same time with the same amount of a given mRNA. A and B) Secretion of SEAP by oocytes, each of which was injected with either 5 ng or 10 ng of SEAP/mRNA. The two groups of oocytes were taken from individual frogs. C) Expression of L-leucine uptake by oocytes each injected with 33 ng of rat kidney mRNA. The uptake of L-leucine by control (water-injected) (6 pmol per oocyte) oocytes has been subtracted. Measurements were made on individual oocytes 48 h after injection of the mRNAs. Each point represents one oocyte.
SECRETED ALKALINE
PHOSPHATASE
In these studies, the first measurements of the proteins expressed from exogenous mRNAs were generally made 20-24 h after injection. At this time, appreciable SEAP was already in the medium.4 SEAP continued to accumulate in the medium during the entire period of the experiment, generally up to 72 h after injection. SEAP secreted by oocytes injected with the same amount of SEAP/mRNA varied considerably among oocytes isolated from the same Xenopus. Thus, in one experiment in which 5 ng of SEAP/mRNA was injected per oocyte, SEAP in the medium after 48 h varied from as little as 5 milliunits to as much as 62 milliunits (average about 34 milliunits SEAP/oocyte) (Fig. 1A). In a similar experiment in which 10 ng of SEAP/mRNA was injected per oocyte, the values of SEAP ranged from about 8 to 150 milliunits (average about 85 milliunits) (Fig. 1B). In both experiments, about 20% of the oocytes failed to secrete detectable amounts of SEAP. Similarly, in oocytes injected with rat kidney mRNA, the levels of expression of Na-independent L-leucine uptake varied over a wide range. Thus, in oocytes injected with 33 ng of the kidney mRNA, the uptake of L-leucine after 48 h varied from about 5 pmol to as much as 65 pmol of L-leucine/(oocyte. 10 mm) (Fig. 1C). Again, about one-fifth of the injected oocytes failed to express Lleucine uptake significantly above that observed in controls (H20 injected) oocytes. No SEAP activity was de-
4When monitoring the expression of proteins with a high turnover rate, it might be desirable to assay SEAP in the medium at earlier times after the injection of mRNAs. In such situations, depending on the amount of SEAP/mRNA injected, either the colonmetric assay or a highly sensitive, bioluminescence-based assay (11) for AP can be used. Indeed, monitoring SEAP in the medium should also facilitate the kinetics of expression of secretory and membrane proteins.
229
tected in the media of either the control or the kidney mRNA-injected oocytes. Also, the oocytes injected with SEAP/mRNA alone exhibited L-leucine uptake similar to that seen in control oocytes, about 5-7 pmol of Lieucine/(oocyte. 10 mm). When SEAP and rat kidney mRNA (3 and 33 ng, respectively) were coinjected into oocytes (Fig. 2), a good correlation was observed between the amount of SEAP and transport of L-leucine (correlation coefficient 0.84). Thus, oocytes that secreted relatively high levels of SEAP also exhibited high expression of L-leucine uptake and vice versa. A similar range of L-ieucine uptake and of SEAP was seen among oocytes injected with either rat kidney mRNA or SEAP/mRNA alone (Fig. 1), indicating that at the amounts used in Fig. 2, when SEAP and kidney mRNAs are coinjected, they do not influence each other’s expression. Figure 2 also shows that expression of -y-GT correlates well with SEAP in oocytes coinjected with SEAP and kidney mRNA. In control oocytes and in oocytes injected with SEAP/ mRNA alone, no y-GT was detected. In a separate experiment, each oocyte in the group was coinjected with 1.5 ng of SEAP/mRNA and 25 ng of kidney mRNA. Here again a good correlation was observed between SEAP and L-leucine uptake (correlation coefficient = 0.93) and between SEAP and -y-GT. =
I
I
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40
SEAP in medium (munits) Figure 2. Correlation between SEAP activity in the medium and L-leucine uptake by individual oocytes coinjected with a mixture of SEAP/mRNA and rat kidney mRNA (3 ng and 33 ng, respectively, per oocyte) (filled circles). Measurements were made 72 h after injection. The open circle denotes the average value for L-leucine uptake by control (water-injected) oocytes. Bars show the average glutamyl transpeptidase (‘y-GT) activity in oocytes coinjected with kidney and SEAP mRNA. The horizontal reach of the bar denotes the range of SEAP secreted by individual oocytes. For y-GT, four oocytes expressing similar levels of SEAP were pooled and assayed as described in Materials and Methods. Each bar represents the average of two such determinations (eight oocytes). The shaded area highlights the correlation between SEAP and the expression of L-leucine transport.
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DISCUSSION The relative stability, high substrate turnover number, and high affinity for a range of substrates have made AP a widely used reagent in many diagnostic and biochemical procedures. These properties help make AP a useful indicator of expression in the Xenopus oocyte system (and, presumably, in other expression systems). Since, like most other cells, oocytes have significant amounts of cell-surface AP (unpublished data), use of the wild-type (membrane-bound) enzyme has limited usefulness as an indicator of expression of foreign mRNAs. To overcome this problem, we used the mRNA encoding a secreted mutant form of human placental alkaline phosphatase. The cDNA encoding SEAP was cloned into a vector that allows large-scale synthesis of SEAP/mRNA in vitro.5 This mutant form of AP is secreted by the oocytes into the medium, thus allowing rapid identification of oocytes that efficiently express the injected mRNAs. An advantage of using the placental form of AP is that, in contrast to other isozymes (including the oocyte enzyme), it is unaffected by 10 mM homoarginine (11). Thus, inclusion of homoarginine in assay media eliminates the possibility of interference by endogenous forms of AP. Injection of as little as 1 ng of SEAP/mRNA per oocyte resulted in the secretion of readily measurable amounts of SEAP into the medium. Using Na-independent L-leucine transport and y-GT (both expressed in oocytes from rat kidney mRNA), we have shown that expression of these two membrane-associated functions and secretion of SEAP parallel each other in oocytes injected with the two mRNAs. The small amounts of SEAP/mRNA used do not interfere with the expression of the test mRNAs SEAP is secreted
-
5
230
In this experiment, SEAP in the medium from individual oocytes varied from about 2 to 27 milliunits, and L-leucine uptake for the corresponding oocytes ranged from 8 to 70 pmol/(oocyte. 10 mm); y-GT activity varied from about 0.2 milliunits per oocyte for the low SEAP expressors to about 2 milliunits per oocyte for the high expressors of SEAP.
Feb. 1990
and the vice medium versa. Furthermore, into and easily
since quan-
tified, each oocyte is available for assay of proteins expressed either on the oocyte surface or intracellularly. SEAP in the medium can be assayed by using standard colorimetric procedures and a number of readily available scanning instruments. For most purposes, however, visual examination of the assay mixtures allows rapid selection of those oocytes that efficiently express the injected mRNAs. Indeed, in our investigations of the cloning and characterization of the mammalian amino acid transporters, we now routinely coinject SEAP/mRNA with the test mRNA (either cellular or that synthesized in vitro from cDNA libraries) and use a visual test for SEAP activity in the medium to quickly
5The vector, pGEM-4Z/PLAP489, be provided to investigators upon
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containing SEAP/cDNA request to S. U.
will
TATE ET AL.
select oocytes for amino acid uptake assay (usually those expressing SEAP above the median). The inherent difficulties usually encountered in the use of the oocyte system, viz, the large variations in expression of foreign mRNAs by individual oocytes from the same animal and among batches from different animals, are thus overcome. The reduction in the number of oocytes that need to be assayed results in an enormous saving of time and expense. Since injection of too much mRNA or poor quality mRNA suppresses translation of both endogenous and exogenous mRNAs, diminished expression of SEAP/mRNA under these conditions signals the investigator to potential problems. SEAP/mRNA will be particularly valuable as a predictive internal indicator of the expression of exogenous mRNAs in oocytes in investigations that involve complex measurements (e.g., electrophysiological, binding assays, etc.) on individual or pooled oocytes. In such experiments, the investigator should select only those oocytes that express the highest amounts of SEAP (e.g., upper 10-20%). SEAP/mRNA will also be useful in other expression systems such as those that use sea urchin eggs and newt (Cynops) oocytes. Recently, a method for the introduction of RNA into tissue culture cells using cationic iiposomes (lipofectin) was introduced (18). This procedure allows RNA transfection of a wide variety of mammalian, amphibian, and insect cells and provides a new approach to the study of factors that influence the translation and stability of eukaryotic mRNAs. SEAP/mRNA may also prove to be a reliable indicator of the efficiency of this transfection process. Thus, we anticipate many uses for SEAP/ mRNA as an internal standard in studies involving expression of eukaryotic mRNAs.
3. 4. 5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16.
REFERENCES 1. Gurdon, oocytes 370-386 2. Colman, Xenopus
J. B., and Wickens, M. P. (1983) The use of Xenopus for the expression of cloned genes. Methods EnzymoL 101,
17. 18.
A. (1984) Translation oocytes. Transcription
of eukaryotic and Translation:
messenger A Practical
RNA in Approach
(Hames, B. D., and Higgins, S. J., eds) pp. 271-300, IRL Press, Washington, DC Melton, D. A. (1987) Translation of messenger RNA in injected frog oocytes. Methods Enzymol. 152, 288-296 Snutch, T. P. (1988) The use of Xenopus oocytes to probe synaptic communication. Trends Neurosci. 11, 250-256 Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., and Nakanishi, S. (1987) cDNA cloning of bovine substance K receptor through oocyte expression system. Nature (London) 329, 836-838 Hediger, M. A., Coady, M. J., Ikeda, T. S., and Wright, E. M. (1987) Expression cloning and cDNA sequencing of Na/ glucose co-transporter. Nature (London) 330, 379-381 Lubbert, H., Hoffmann, B. J., Snutch, T. P., Van Dyke, T., Levine, A. J., Hartig, P. R., Lester, H. A., and Davidson, N. (1987) cDNA cloning of a serotonin 5-HT,c receptor by dcctrophysiological assays of mRNA-injected Xenopus oocytes. Proc. NatI. Acad. Sd. USA 84, 4332-4336 Julius, D., MacDermott, A. B., Axel, R., and Jessell, T. M. (1988) Molecular characterization of a functional cDNA encoding the serotonin Ic receptor. Science 241, 558-564 Takumi, T., Ohkubo, H., and Nakanishi, 5. (1988) Cloning of a membrane protein that induces a slow voltage-gated potassium current. Science 242, 1042-1045 Bergen, J., Hauber, J., Hauber, R., Geiger, R., and Cullen, B. R. (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66, 1-10 Berger,J., Howard, A. D., Gerger, L., Cullen, B. R., and Udenfriend, 5. (1987) Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. Proc. NatI. Acad. Sd. USA 84, 4885-4889 Chomczynski, P., and Sacchi, N. (1987) Single-step method for RNA isolation by acid guanidium thiocyanate-phenol-chioroform extraction. AnaL Biochem. 162, 156-159 Badley, J. E., Bishop, G. A., St. John, T., and Frelinger, J. A. (1988) A simple, rapid method for the purification of poly A RNA. Biotechniques 6, 114-116 Wallace, R. A., Jared, D. W., Dumont, J. N., and Sega, M. W. (1973) Protein incorporation by isolated amphibian oocytes: optimum incubation conditions. j Exp. Zool. 184, 321-334 Christensen, H. N. (1985) On the strategy of kinetic discrimination of amino acid transport systems. J. Membr. BioL 84, 9 7-103 Tate, S. S., Urade, R., Getchell, T. V., and Udenfniend, S. (1989) Expression of the mammalian Na-independent L system amino acid transporter in Xenopus laevis oocytes. Arch. Biochem. Biophys. 275, 591-596 Tate, S. S., and Meister, A. (1985) ‘y-Glutamyl transpeptidase from kidney. Methods Enzymol. 113, 400-419 Malone, R. W., Felgner, P. L., and Verma, I. M. (1989) Cationic liposome-mediated RNA transfection. Proc. Natl. Acad. Sci. USA 86, 6077-6081
Receivedfor publication October 26, 1989. Acceptedfor publication November 22, 1989.
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PHOSPHATASE
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