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Dot assay for neomycin phosphotransferase activity in crude cell extracts
ANALYTICAL
BIOCHEMISTRY
162,52%535
(1987)
Dot Assay for Neomycin Phosphotransferase STEVEN G. PLATT’
Activity in Crude Cell Extracts
AND NINGSUN
Agracetus, 8520 University Green, Middleton,
YANG Wisconsin 53562
Received June 20, I986 A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assayemploys a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not. o 1987Academic P-, IX KEY WORDS: aminoglycoside phosphotransferase; assay of gene expression; gene transfer; neomycin phosphotransferase; protein kinase; recombinant DNA.
Aminoglycoside antibiotic resistance resulting from neomycin phosphotransferase (NPT II)’ activity is widely used as a dominant selectable marker for the isolation of transformed cells resulting from gene transformation experiments (l-7). However, the use of NPT II as a reporter gene relative to that of chloramphenicol acetyltransferase (CAT) in studies of foreign gene expression in eukaryotic systems has been limited. This is likely due to the lack of a convenient NPT II activity assay. Development of such an assay would allow NPT II to be used as both a selectable marker and a reporter gene. CAT has not been shown to be useful for selection in most eukaryotic systems. The current procedure for assaying NPT II activity in eukaryotic tissue is unsuitable for the rapid ’ Present address: Fairview Industries, 8616 Fairway Place, Middleton, WI 53562. 2 Abbreviations used: NPT II, neomycin phosphotransferase; CAT, chloramphenicol acetyltransferase; PK, protein kinase; PAGE, polyacrylamide gel electrophoresis.
529
screening of the large number of transformed cell cultures produced in plant and animal genetic engineering experiments. It includes a gel electrophoretic separation of the active enzyme (6-9) followed by indicator gel overlay assay using kanamycin or neomycin and labeled ATP as substrates. The protein separation step is required in order to eliminate positive interference resulting from the presence of high levels of protein kinase (PK)like activities in eukaryotic crude cell extracts. The enzymatic activities of both PK and NPT II produce 32P-labeled products, phosphorylated proteins and kanamycin phosphate, respectively, that bind to P81 phosphocellulose paper in the final step of the assay (8,9). Only bound labeled kanamytin phosphate ( 10) is a measure of the NPT II activity. We now describe a dot assay that is based on the elimination of positive interference by passing extracts incubated with NPT II substrates through stacked nitrocellulose (NC) and P81 ion-exchange papers. The procedure obviates the need for prior electro-
0003-2697187 $3.00 Copyright Q 1987 by Academic Press, Inc. AU rights of reproduction in any form reserved.
530
PLATT
AND
phoretic separation in detecting NPT II activity in crude plant extracts. MATERIALS
AND METHODS
Chemicals and adsorption papers. Kanamycin and neomycin sulfates were obtained from Sigma. P8 1 phosphocellulose paper was obtained from Whatman and nitrocellulose paper (0.45 pm) from Schleicher & Schuell. Neomycin phosphotransferase II purified from Escherichia coli was a generous gilt of Dr. David Gelfand, Cetus Corp. Transformed cells. Ti plasmid vectors were constructed with the Tn5 encoded NPT II gene, and plant cell transformations were carried out by infection with the vector host, Agrobacterium tumefaciens. Transformed plant callus tissues of tobacco (Nicotiana tabacum cv. Havana 425) and cotton (Gossypium hirsutum cvs. Delta Pine 6 1, Stoneville 213 and Coker 3 12) were selected on MS media (11) containing 75-100 I.cg/ml of kanamycin. E. coli cells carrying an NPT II expression plasmid were also used in this experiment. Crude cell extract preparation. Calli were preincubated with buffer containing 30 mM NaCl, 15 mM NH&l, 3 mM MgC12, 5.5 M Na2EDTA, 2.3 mM Tris, 0.2 mM DDT, 105 mM sucrose, pH 7.0. Following freeze-thaw (liquid Nz, 37”C), tissues were ground and sonicated in buffer containing 37 mM Na2EDTA, 126 mM NaCl, 83 mM NH&l, 7.7 mM Tris, 15 mM DTT, 2.5 mM phenylmethylsulfonyl fluoride, 0.6 mg/ml leupeptin, 1.05 mg/ml soybean trypsin inhibitor, and 5.0 mg/ml bovine serum albumin. The last four reagents were added to protect against protease degradation of NPT II. Forty microliters of extraction buffer was used per 100 mg of callus tissue (fresh wt). Supematants were collected following microfuge centrifugation. E. coli cell extracts were produced by sonication in extraction buffer.
YANG
Electrophoretic analysis of NPT II activity. The procedure was based on that developed by Reiss et al. (7) for animal and bacterial cell extracts and modified for plant cell lysates by Paszkowski et al. (6). Extract (lo-40 ~1) was applied to a nondenaturing polyacrylamide gel (separating gel: 7.8% acrylamide, 0.26% bisacrylamide (bis), 0.38 M Tris, pH 8.8; stacking gel: 4.4% acrylamide, 0.15% bis, 0.13 M Tris, pH 6.8; running buffer: 38 mM glycine, 5 mM Tris, pH 8.8). Following electrophoresis (4 h, 18 V/cm) the separating gel was exchanged against buffer No. 1 (67 mM Tris-maleate, 42 mM MgCl,, 400 mM NH&l, pH 7.1). The separating gel was then overlayered on a 1% low-meltingpoint agarose gel containing 50-100 &i[r32P]ATP (ca. 5000 Ci/mmol), kanamycin, or neomycin sulfate (26 hg/ml of the free base) and equal volumes of water and buffer No. 1, and incubated for 2.5 h at 27°C. The agarose gel was then blotted (12 h) through P8 1 paper (conventional procedure) or a single sheet of nitrocellulose paper followed by P8 1 paper. Papers were washed in warm water to remove unreacted ATP, dried, and exposed to X-ray film (type XAR-5, Kodak Corp.), and radioactivity was quantified by scintillation counting. A factor for calculating NPT II activity (cpm/ng) present was determined for each gel by including two lanes with a known amount of purified NPT II. Dot assay of NPT II activity. A 1 IO+1 aliquot of buffer No. 1 containing kanamycin or neomycin sulfate (39 pg/ml of the freebase), a 20-~1 aliquot of sample extract containing 5-10 pg plant proteins, and then 50 ~1 of [T-~*P]ATP in buffer No. 1 (750,000 cpm, ca. 5000 Ci/mmol) were mixed in the wells of a 96-place microwell plate. In some cases duplicate samples containing no antibiotic were prepared. After 2f h, the reaction solutions were transferred into the wells of a microsample filtration manifold (Schleicher & Schuell Minifold) fitted with a layer of NC paper above a folded double layer of P81 paper and a single sheet of Whatman 3MM
DOT NEOMYCIN
PHOSPHOTRANSFERASE
paper. All papers were presaturated with distilled water. After the wells had drained (gravity flow, 1 h), each well was washed with three 250~~1 aliquots of deionized water and drained by mild suction. The NC and P81 filter papers were then removed, further washed with deionized water, dried, and exposed to X-ray film for ca. l-4 h. Dot radioactivity was determined by scintillation counting. To quantify NPT II activity in crude plant extracts, results for a series of cotton extract unknowns were normalized using duplicate samples to which 5 ng of purified E. coli NPT II was added as an internal standard. This was done to correct the interference of assay activity caused by inhibitors of NPT II, which are present in crude plant extracts (see Results and Discussion). A factor for the radioactivity per nanogram of NPT II in a sample in the presence of kanamycin was determined from the expression (cpm, +standard) - (cpm, -standard)/ 5; following a background (58 cpm) correction, the value for (cpm, -standard) was divided by that factor to determine the test sample’s NPT II contents. RESULTS AND DISCUSSION
Nitrocellulose adsorption of protein kinase-like products. An identical series of extracts of tobacco calli, as well as an extract of NPT II gene-transformed E. coli, was applied to each half of a PAGE separating gel. On a second gel, extracts of three transformed cotton calli were applied to each half of gel. Following electrophoresis both gels were exposed to indicator gel containing [32P]ATP and kanamycin. The left half of each indicator gel was then blotted through P8 1 paper as in a conventional assay. The right half was blotted through NC paper placed in between the indicator gel and the P8 I paper (Fig. 1). On the left half of each gel, radioactive bands resulting from NPT II and PK-like activity were both absorbed on the P8 1 paper. On the right half, bands resulting from PK-like ac-
ASSAY
531
tivity were adsorbed on the NC paper only, whereas kanamycin phosphate resulting from NPT II activity passed through and was adsorbed on the P81 paper. The identity of the band resulting from NPT II activity was confirmed by its kanamycin dependence and also by comparison of its location to that produced by purified NPT II. In separate experiments, Western blot analysis using antibody against NPT II confirmed the presence of NPT II at that location in the separating gel (data not shown). Extracts of control (nontransformed) calli contain PK-like activity but no activity in the region of NPT II (Fig. IA, lanes 1 and 9). It is conceivable that, in certain host cells, PK-like enzymes might migrate with the same mobility as NPT II because of the presence of high-mobility PK-like enzymes or with decreased NPT II mobility resulting from processing (e.g., glycosylation). In such cases, the NC/P8 1 double blotting technique distinguishes authentic NPT II activity from PKlike activity. The NC blot itself provides the basis for an assay useful in studies of protein kinase activity. Additional experimentation has indicated that kanamycin inhibits some plant PK-like enzymes present in cotton and soybean extracts and also that those bands most likely result from protein self-phosphorylation (to be published).
Dot method analysis of solutions containing NPT II and extracts ofplant tissues.Several types of samples were assayed including serial dilutions of purified NPT II in buffer and control (untransformed) tobacco tissue extracts and extracts of control and transformed calli (confirmed by gel analysis of NPT II activity). Analyses were conducted in the presence or absence of kanamycin (Fig. 2). In the cases of NPT II dilutions in buffer and in control callus extract, substantial P8 1 -bound 32P radioactivity was found only in the presence of kanamycin and shows a linear response to the NPT II activity present. No activity was found on the NC paper when NPT II was diluted in buffer.
532
PLATT
AND YANG
P8I
NC
P8l
(A) Origin
PK-like Activity I
NPTII
Lane Paper
I23
4
5 NC
P8I
6
4
5 P8l
6
(N
FIG. 1. Separation of electrophoretically banded protein k&se-like and NPT II activities by blotting on agarose indicator gel through nitrocellulose and P81 papers. (A) Lanes 1 and 9, extract from untransformed tobacco callus; lanes 2-5 and 10-l 3, extract from transformed tobacco callus; lanes 6 and 14 were empty; lanes 7 and 15, extract from transformed E. coli. (B) All lanes contained extract from transformed cotton calli.
The 32P-labeling activity was readily detectable on the NC paper when both control and transformed calli were assayed. Significant P8 1-bound radioactivity was present only when NPT II-producing &us cultures were assayed and added kanamycin was present. These results are consistent with the gel analysis data presented in Fig. 1. Therefore, the combination of NC and P81 ion adsorption papers distinguished between the products of PK-like and NPT II enzymatic activities in this dot assay. When extracts of transformed calli were examined, the radioactivity adsorbed on the P81 paper in the absence of added kanamycin averaged 7% of that ab-
sorbed in the presence of added antibiotic. This activity detected in the absence of exogenously added antibiotic may result from the presence of low levels of kanamycin in the caIli resulting from the transformant selection process. Sensitivity of the dot method. The method could easily detect 0.01 ng of NPT II in buffer (Fig. 3). With plant callus extract the method was sensitive to 0.1 ng (Fig. 4). These results show that there is a IO-fold reduction in assay sensitivity when plant extract, instead of buffer alone, is used in the dot method. This appears to be caused by the presence of certain inhibitors of NPT II
DOT NEOMYCIN
~iL~“~P:IIIUFF~R
-- +tcw
-iuN
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WK
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PHOSPHOTRANSFERASE
fiLH%U~P!~‘ZOYTROL TOBACCO CALLUS EXlRACT -WIN
--+t$N p81 NC
-ivd P8l
NC
:
s
J
Tf
FIG.
TOBACCO CALLUS EXTRACT +KN -__ P81 NC
F%l K
533
ASSAY
2. Dot assayfor NPT II activity; aliquots of 20 ~1 tobacco callus extracts were tested.
present in plant extracts. An experiment was performed to determine the sensitivity and inhibition of the dot assay as a function of the amount of total protein in the extract. Table 1 shows that inhibitors in plant extracts containing 5 to 10 pg total protein re-
sulted in a lo- to 15-fold reduction in NPT II activity in test samples. We also found that when a known amount of E. coli NPT II enzyme (0.1 to 5 ng) in extraction buffer was assayed, the dot procedure resulted in a recovery of 14.2 f 4.3-fold more radioactivity than that normally obtained by the conventional gel assay (data from three experiments, n = 9). For example, 1 ng NPT II
I
01
Neomycin
IO
PhosphotronsferaseIC,
100
ng
1.00
0.10 0.01
0.04
0.15 0.6 ng NPT II
2.5
10.0
FIG. 3. Dot assayof purified NPT II activity; samples with varying amounts of NPT II were dissolved in extraction buffer.
Neomycin
Phosphotransferose
II, ng
4. Dot assayfor NPT II activity in the presence of extract from untransformed cotton callus; aliquots of 20 ~41of cotton callus extracts containing 3 to 10 pg protein were applied for each sample. FIG.
534
PLATT TABLE
AND YANG
1
INHIBITORYEFFECTOFTOBACCOCALLUSEXTRACT ON m
11 DOT
&SAY
Total proteins in plant extracts (PIi!)
Relative NPT II activity (9%)’
0 (control) 0.5 2 5 10
100 31.2 17.1 11.6 7.2
LIDuplicate samples of increasing amounts of tobacco extracts were added to 5 ng of NPT II, and enzyme activity was determined by standard dot assay. A radioactivity of 43,045 cpm/ng was obtained from control samples where no plant extract was added, and this value, as 100% activity, was used as standard for obtaining relative activity of other test samples.
assayed with the dot and gel methods gave an activity of 59,400 and 3423 cpm, respectively. This is presumably due to the fact that enzymatic reaction is more efficient in liquid (dot assay) than in the gel-to-gel contact phase (gel assay). Therefore, even though there is a lo- to 15-fold reduction in activity when cell extracts containing inhibitors are present in the dot assay, an average 1Cfold increase in recoverable activity (compared to the gel assay) effectively results in the sensitivity of the dot assay being similar to that of the gel assay. Under these conditions, the standard dot assay detected 0.1 ng NPT II in plant extracts containing 5 to 10 pg proteins. The reported sensitivity of gel method is 0.1 ng of enzyme (7), and this is in good agreement with our results using both gel and dot assay methods. The production of labeled aminoglycoside phosphate in both assays can be increased by using neomycin instead of kanamycin as a substrate: enzymatic activity increased by a factor of 4.2 + 1.2 (n = 5) in the gel assay and by 1.6 f 0.3 (n = 6) in the dot assay.
Correlation between NPT II determinations by the dot and gel methods. The NPT II
level of each of a series of samples of putatively transformed cotton calli was assayed using both methods (Fig. 5). The correlation coefficient of the logs of the activity values obtained by the two methods was 0.83 (n = 66). Results in Fig. 5 shows that the dot method provides an effective means for screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not. CONCLUSIONS
The dot assay described herein provides a convenient and rapid procedure for analyzing crude cell extracts for the presence of NPT II. It allows an order of magnitude increase in the number of samples manageable by a researcher in a given period of time, provides for assay completion in a single day, and is comparable in sensitivity to the conventional electrophoretic procedure. Assay results obtained correlate well with those of the conventional procedure. Thus, the dot assay can be used for screening large numbers of cell cultures for high level NPT II
100 1
O”I:: 001 0.1
IO
100
Gel: NPT It (ng)
FIG. 5. Comparison of NPT II determinations on a series of putatively transformed cotton calli using the gel and dot methods. Values were calculated as described under Materials and Methods.
DOT NEOMYCIN
PHOSPHOTRANSFERASE
producers and presumably for high level expression of additional foreign genes constructed in tandem to NPT II in transformed cells. The convenience of this dot assay method for NFT II and the fact that it serves as a dominant, selectable marker in most tested eukaryotic tissue culture systems suggest that NPT II gene can be used more effectively than CAT gene as a reporter gene for genetic transformation experiments in eukaryotic systems. ACKNOWLEDGMENTS We thank Kenneth Barton for vector construction and helpful conversations; Barry Cohen, Gail Johnson, Heidi Poser, and Paul Umbeck for tissue culture manip ulations; David Gelfand for purified NPT II; and Joseph Burkholder for excellent technical assistance.
REFERENCES 1. An, G., Watson, B. D., Stachel, S., Gordon, M. P., and Nester, E. W. (1985) EMBO J. 4,217-284. 2. Colbere-Garapin, F., Horodniceanu, F., Kourilsky, P., and Garapin, A.-C. (1981) J. Mol. Biol. 150, l-14.
ASSAY
535
3. De Block, M., Herrera-Estrella, L., Van Montagu, M., Schell, J., and Zambryski, P. ( 1984) EMBO J. 3, 1681-1689. 4. Franke, C. A., Rice, C. M., Strauss, J. H., and Hruby, D. E. (1985) Mol. Cell Biol. 5, 1918-1924. 5. Herrera-Estrella, L., De Block, M., Messens, E., Hernalsteens, J.-P., Van Montagu, M., and Schell, J. (1983) EMBO J. 2,987-995. 6. Paszkowski, J., Shillito, R. D., Saul, M., Mandak, V., Hohn, T., Hohn, B., and Potrykus, I. (1984) EMBO J. 3,2111-2122. 7. Reiss, B., Sprengel, R., Will, H., and Schaller, H. (1984) Gene30,21 I-218. 8. Schreier, P. H., Seftor, E. A., Schell, J., and Bohnert, H. J. ( 1985) EM30 J.4,25-32. 9. Fregien, N., and Davidson, N. (1985) Anal. Biothem. 148, 101-104. 10. Van den Broeck, G., Timko, M. P., Kausch, A. P., Cashmore, A. R., Van Montagu, M., and Herrera-Estrella, L. (1985) Nature (London) 313, 358-363.
11. Murashigue, T., and Skoog, R. (1962) Physiol. Plant. l&413-497. 12. Kiselev, V. I., Gorkun, A. F., and Rechinskii, V. 0. (1985) Anal. B&hem. 146,434-436.
BIOCHEMISTRY
162,52%535
(1987)
Dot Assay for Neomycin Phosphotransferase STEVEN G. PLATT’
Activity in Crude Cell Extracts
AND NINGSUN
Agracetus, 8520 University Green, Middleton,
YANG Wisconsin 53562
Received June 20, I986 A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assayemploys a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not. o 1987Academic P-, IX KEY WORDS: aminoglycoside phosphotransferase; assay of gene expression; gene transfer; neomycin phosphotransferase; protein kinase; recombinant DNA.
Aminoglycoside antibiotic resistance resulting from neomycin phosphotransferase (NPT II)’ activity is widely used as a dominant selectable marker for the isolation of transformed cells resulting from gene transformation experiments (l-7). However, the use of NPT II as a reporter gene relative to that of chloramphenicol acetyltransferase (CAT) in studies of foreign gene expression in eukaryotic systems has been limited. This is likely due to the lack of a convenient NPT II activity assay. Development of such an assay would allow NPT II to be used as both a selectable marker and a reporter gene. CAT has not been shown to be useful for selection in most eukaryotic systems. The current procedure for assaying NPT II activity in eukaryotic tissue is unsuitable for the rapid ’ Present address: Fairview Industries, 8616 Fairway Place, Middleton, WI 53562. 2 Abbreviations used: NPT II, neomycin phosphotransferase; CAT, chloramphenicol acetyltransferase; PK, protein kinase; PAGE, polyacrylamide gel electrophoresis.
529
screening of the large number of transformed cell cultures produced in plant and animal genetic engineering experiments. It includes a gel electrophoretic separation of the active enzyme (6-9) followed by indicator gel overlay assay using kanamycin or neomycin and labeled ATP as substrates. The protein separation step is required in order to eliminate positive interference resulting from the presence of high levels of protein kinase (PK)like activities in eukaryotic crude cell extracts. The enzymatic activities of both PK and NPT II produce 32P-labeled products, phosphorylated proteins and kanamycin phosphate, respectively, that bind to P81 phosphocellulose paper in the final step of the assay (8,9). Only bound labeled kanamytin phosphate ( 10) is a measure of the NPT II activity. We now describe a dot assay that is based on the elimination of positive interference by passing extracts incubated with NPT II substrates through stacked nitrocellulose (NC) and P81 ion-exchange papers. The procedure obviates the need for prior electro-
0003-2697187 $3.00 Copyright Q 1987 by Academic Press, Inc. AU rights of reproduction in any form reserved.
530
PLATT
AND
phoretic separation in detecting NPT II activity in crude plant extracts. MATERIALS
AND METHODS
Chemicals and adsorption papers. Kanamycin and neomycin sulfates were obtained from Sigma. P8 1 phosphocellulose paper was obtained from Whatman and nitrocellulose paper (0.45 pm) from Schleicher & Schuell. Neomycin phosphotransferase II purified from Escherichia coli was a generous gilt of Dr. David Gelfand, Cetus Corp. Transformed cells. Ti plasmid vectors were constructed with the Tn5 encoded NPT II gene, and plant cell transformations were carried out by infection with the vector host, Agrobacterium tumefaciens. Transformed plant callus tissues of tobacco (Nicotiana tabacum cv. Havana 425) and cotton (Gossypium hirsutum cvs. Delta Pine 6 1, Stoneville 213 and Coker 3 12) were selected on MS media (11) containing 75-100 I.cg/ml of kanamycin. E. coli cells carrying an NPT II expression plasmid were also used in this experiment. Crude cell extract preparation. Calli were preincubated with buffer containing 30 mM NaCl, 15 mM NH&l, 3 mM MgC12, 5.5 M Na2EDTA, 2.3 mM Tris, 0.2 mM DDT, 105 mM sucrose, pH 7.0. Following freeze-thaw (liquid Nz, 37”C), tissues were ground and sonicated in buffer containing 37 mM Na2EDTA, 126 mM NaCl, 83 mM NH&l, 7.7 mM Tris, 15 mM DTT, 2.5 mM phenylmethylsulfonyl fluoride, 0.6 mg/ml leupeptin, 1.05 mg/ml soybean trypsin inhibitor, and 5.0 mg/ml bovine serum albumin. The last four reagents were added to protect against protease degradation of NPT II. Forty microliters of extraction buffer was used per 100 mg of callus tissue (fresh wt). Supematants were collected following microfuge centrifugation. E. coli cell extracts were produced by sonication in extraction buffer.
YANG
Electrophoretic analysis of NPT II activity. The procedure was based on that developed by Reiss et al. (7) for animal and bacterial cell extracts and modified for plant cell lysates by Paszkowski et al. (6). Extract (lo-40 ~1) was applied to a nondenaturing polyacrylamide gel (separating gel: 7.8% acrylamide, 0.26% bisacrylamide (bis), 0.38 M Tris, pH 8.8; stacking gel: 4.4% acrylamide, 0.15% bis, 0.13 M Tris, pH 6.8; running buffer: 38 mM glycine, 5 mM Tris, pH 8.8). Following electrophoresis (4 h, 18 V/cm) the separating gel was exchanged against buffer No. 1 (67 mM Tris-maleate, 42 mM MgCl,, 400 mM NH&l, pH 7.1). The separating gel was then overlayered on a 1% low-meltingpoint agarose gel containing 50-100 &i[r32P]ATP (ca. 5000 Ci/mmol), kanamycin, or neomycin sulfate (26 hg/ml of the free base) and equal volumes of water and buffer No. 1, and incubated for 2.5 h at 27°C. The agarose gel was then blotted (12 h) through P8 1 paper (conventional procedure) or a single sheet of nitrocellulose paper followed by P8 1 paper. Papers were washed in warm water to remove unreacted ATP, dried, and exposed to X-ray film (type XAR-5, Kodak Corp.), and radioactivity was quantified by scintillation counting. A factor for calculating NPT II activity (cpm/ng) present was determined for each gel by including two lanes with a known amount of purified NPT II. Dot assay of NPT II activity. A 1 IO+1 aliquot of buffer No. 1 containing kanamycin or neomycin sulfate (39 pg/ml of the freebase), a 20-~1 aliquot of sample extract containing 5-10 pg plant proteins, and then 50 ~1 of [T-~*P]ATP in buffer No. 1 (750,000 cpm, ca. 5000 Ci/mmol) were mixed in the wells of a 96-place microwell plate. In some cases duplicate samples containing no antibiotic were prepared. After 2f h, the reaction solutions were transferred into the wells of a microsample filtration manifold (Schleicher & Schuell Minifold) fitted with a layer of NC paper above a folded double layer of P81 paper and a single sheet of Whatman 3MM
DOT NEOMYCIN
PHOSPHOTRANSFERASE
paper. All papers were presaturated with distilled water. After the wells had drained (gravity flow, 1 h), each well was washed with three 250~~1 aliquots of deionized water and drained by mild suction. The NC and P81 filter papers were then removed, further washed with deionized water, dried, and exposed to X-ray film for ca. l-4 h. Dot radioactivity was determined by scintillation counting. To quantify NPT II activity in crude plant extracts, results for a series of cotton extract unknowns were normalized using duplicate samples to which 5 ng of purified E. coli NPT II was added as an internal standard. This was done to correct the interference of assay activity caused by inhibitors of NPT II, which are present in crude plant extracts (see Results and Discussion). A factor for the radioactivity per nanogram of NPT II in a sample in the presence of kanamycin was determined from the expression (cpm, +standard) - (cpm, -standard)/ 5; following a background (58 cpm) correction, the value for (cpm, -standard) was divided by that factor to determine the test sample’s NPT II contents. RESULTS AND DISCUSSION
Nitrocellulose adsorption of protein kinase-like products. An identical series of extracts of tobacco calli, as well as an extract of NPT II gene-transformed E. coli, was applied to each half of a PAGE separating gel. On a second gel, extracts of three transformed cotton calli were applied to each half of gel. Following electrophoresis both gels were exposed to indicator gel containing [32P]ATP and kanamycin. The left half of each indicator gel was then blotted through P8 1 paper as in a conventional assay. The right half was blotted through NC paper placed in between the indicator gel and the P8 I paper (Fig. 1). On the left half of each gel, radioactive bands resulting from NPT II and PK-like activity were both absorbed on the P8 1 paper. On the right half, bands resulting from PK-like ac-
ASSAY
531
tivity were adsorbed on the NC paper only, whereas kanamycin phosphate resulting from NPT II activity passed through and was adsorbed on the P81 paper. The identity of the band resulting from NPT II activity was confirmed by its kanamycin dependence and also by comparison of its location to that produced by purified NPT II. In separate experiments, Western blot analysis using antibody against NPT II confirmed the presence of NPT II at that location in the separating gel (data not shown). Extracts of control (nontransformed) calli contain PK-like activity but no activity in the region of NPT II (Fig. IA, lanes 1 and 9). It is conceivable that, in certain host cells, PK-like enzymes might migrate with the same mobility as NPT II because of the presence of high-mobility PK-like enzymes or with decreased NPT II mobility resulting from processing (e.g., glycosylation). In such cases, the NC/P8 1 double blotting technique distinguishes authentic NPT II activity from PKlike activity. The NC blot itself provides the basis for an assay useful in studies of protein kinase activity. Additional experimentation has indicated that kanamycin inhibits some plant PK-like enzymes present in cotton and soybean extracts and also that those bands most likely result from protein self-phosphorylation (to be published).
Dot method analysis of solutions containing NPT II and extracts ofplant tissues.Several types of samples were assayed including serial dilutions of purified NPT II in buffer and control (untransformed) tobacco tissue extracts and extracts of control and transformed calli (confirmed by gel analysis of NPT II activity). Analyses were conducted in the presence or absence of kanamycin (Fig. 2). In the cases of NPT II dilutions in buffer and in control callus extract, substantial P8 1 -bound 32P radioactivity was found only in the presence of kanamycin and shows a linear response to the NPT II activity present. No activity was found on the NC paper when NPT II was diluted in buffer.
532
PLATT
AND YANG
P8I
NC
P8l
(A) Origin
PK-like Activity I
NPTII
Lane Paper
I23
4
5 NC
P8I
6
4
5 P8l
6
(N
FIG. 1. Separation of electrophoretically banded protein k&se-like and NPT II activities by blotting on agarose indicator gel through nitrocellulose and P81 papers. (A) Lanes 1 and 9, extract from untransformed tobacco callus; lanes 2-5 and 10-l 3, extract from transformed tobacco callus; lanes 6 and 14 were empty; lanes 7 and 15, extract from transformed E. coli. (B) All lanes contained extract from transformed cotton calli.
The 32P-labeling activity was readily detectable on the NC paper when both control and transformed calli were assayed. Significant P8 1-bound radioactivity was present only when NPT II-producing &us cultures were assayed and added kanamycin was present. These results are consistent with the gel analysis data presented in Fig. 1. Therefore, the combination of NC and P81 ion adsorption papers distinguished between the products of PK-like and NPT II enzymatic activities in this dot assay. When extracts of transformed calli were examined, the radioactivity adsorbed on the P81 paper in the absence of added kanamycin averaged 7% of that ab-
sorbed in the presence of added antibiotic. This activity detected in the absence of exogenously added antibiotic may result from the presence of low levels of kanamycin in the caIli resulting from the transformant selection process. Sensitivity of the dot method. The method could easily detect 0.01 ng of NPT II in buffer (Fig. 3). With plant callus extract the method was sensitive to 0.1 ng (Fig. 4). These results show that there is a IO-fold reduction in assay sensitivity when plant extract, instead of buffer alone, is used in the dot method. This appears to be caused by the presence of certain inhibitors of NPT II
DOT NEOMYCIN
~iL~“~P:IIIUFF~R
-- +tcw
-iuN
P81K
WK
c *
PHOSPHOTRANSFERASE
fiLH%U~P!~‘ZOYTROL TOBACCO CALLUS EXlRACT -WIN
--+t$N p81 NC
-ivd P8l
NC
:
s
J
Tf
FIG.
TOBACCO CALLUS EXTRACT +KN -__ P81 NC
F%l K
533
ASSAY
2. Dot assayfor NPT II activity; aliquots of 20 ~1 tobacco callus extracts were tested.
present in plant extracts. An experiment was performed to determine the sensitivity and inhibition of the dot assay as a function of the amount of total protein in the extract. Table 1 shows that inhibitors in plant extracts containing 5 to 10 pg total protein re-
sulted in a lo- to 15-fold reduction in NPT II activity in test samples. We also found that when a known amount of E. coli NPT II enzyme (0.1 to 5 ng) in extraction buffer was assayed, the dot procedure resulted in a recovery of 14.2 f 4.3-fold more radioactivity than that normally obtained by the conventional gel assay (data from three experiments, n = 9). For example, 1 ng NPT II
I
01
Neomycin
IO
PhosphotronsferaseIC,
100
ng
1.00
0.10 0.01
0.04
0.15 0.6 ng NPT II
2.5
10.0
FIG. 3. Dot assayof purified NPT II activity; samples with varying amounts of NPT II were dissolved in extraction buffer.
Neomycin
Phosphotransferose
II, ng
4. Dot assayfor NPT II activity in the presence of extract from untransformed cotton callus; aliquots of 20 ~41of cotton callus extracts containing 3 to 10 pg protein were applied for each sample. FIG.
534
PLATT TABLE
AND YANG
1
INHIBITORYEFFECTOFTOBACCOCALLUSEXTRACT ON m
11 DOT
&SAY
Total proteins in plant extracts (PIi!)
Relative NPT II activity (9%)’
0 (control) 0.5 2 5 10
100 31.2 17.1 11.6 7.2
LIDuplicate samples of increasing amounts of tobacco extracts were added to 5 ng of NPT II, and enzyme activity was determined by standard dot assay. A radioactivity of 43,045 cpm/ng was obtained from control samples where no plant extract was added, and this value, as 100% activity, was used as standard for obtaining relative activity of other test samples.
assayed with the dot and gel methods gave an activity of 59,400 and 3423 cpm, respectively. This is presumably due to the fact that enzymatic reaction is more efficient in liquid (dot assay) than in the gel-to-gel contact phase (gel assay). Therefore, even though there is a lo- to 15-fold reduction in activity when cell extracts containing inhibitors are present in the dot assay, an average 1Cfold increase in recoverable activity (compared to the gel assay) effectively results in the sensitivity of the dot assay being similar to that of the gel assay. Under these conditions, the standard dot assay detected 0.1 ng NPT II in plant extracts containing 5 to 10 pg proteins. The reported sensitivity of gel method is 0.1 ng of enzyme (7), and this is in good agreement with our results using both gel and dot assay methods. The production of labeled aminoglycoside phosphate in both assays can be increased by using neomycin instead of kanamycin as a substrate: enzymatic activity increased by a factor of 4.2 + 1.2 (n = 5) in the gel assay and by 1.6 f 0.3 (n = 6) in the dot assay.
Correlation between NPT II determinations by the dot and gel methods. The NPT II
level of each of a series of samples of putatively transformed cotton calli was assayed using both methods (Fig. 5). The correlation coefficient of the logs of the activity values obtained by the two methods was 0.83 (n = 66). Results in Fig. 5 shows that the dot method provides an effective means for screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not. CONCLUSIONS
The dot assay described herein provides a convenient and rapid procedure for analyzing crude cell extracts for the presence of NPT II. It allows an order of magnitude increase in the number of samples manageable by a researcher in a given period of time, provides for assay completion in a single day, and is comparable in sensitivity to the conventional electrophoretic procedure. Assay results obtained correlate well with those of the conventional procedure. Thus, the dot assay can be used for screening large numbers of cell cultures for high level NPT II
100 1
O”I:: 001 0.1
IO
100
Gel: NPT It (ng)
FIG. 5. Comparison of NPT II determinations on a series of putatively transformed cotton calli using the gel and dot methods. Values were calculated as described under Materials and Methods.
DOT NEOMYCIN
PHOSPHOTRANSFERASE
producers and presumably for high level expression of additional foreign genes constructed in tandem to NPT II in transformed cells. The convenience of this dot assay method for NFT II and the fact that it serves as a dominant, selectable marker in most tested eukaryotic tissue culture systems suggest that NPT II gene can be used more effectively than CAT gene as a reporter gene for genetic transformation experiments in eukaryotic systems. ACKNOWLEDGMENTS We thank Kenneth Barton for vector construction and helpful conversations; Barry Cohen, Gail Johnson, Heidi Poser, and Paul Umbeck for tissue culture manip ulations; David Gelfand for purified NPT II; and Joseph Burkholder for excellent technical assistance.
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ASSAY
535
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