- Email: [email protected]
A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein
ANALYTICAL
109, 3 17-320
BIOCHEMISTRY
(1980)
A Simple Electrophoretic Polypeptide Composition D. Lahorutorium
Procedure for the Determination of the of the Subunits of Fraction 1 Protein’
CAMMAERTS
AND
vow Plantmgenetica. B-1640 St.-Genesius-Rodeo Received
April
M. Vrijr
JACOBS Univrrsiteit Belgium
Bnrssel.
29. 1980
A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 M urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.
Fraction 1 protein (ribulosebiphosphate carboxylase EC 4.1.1.39), the major soluble protein in leaves of higher plants, is composed of large and small subunits (1). The large subunit has been shown to be chloroplast coded and maternally inherited, whereas the small subunit is coded by nuclear DNA (2.3). The polypeptide composition of the subunits of Fraction 1 protein has been determined by isoelectric focusing in 8 M urea using samples of purified carboxymethylated Fraction 1 protein (4,5). Fraction 1 protein was purified either by crystallization (6) or by salt fractionation and column chromatography (5). In the case where no crystalline Fraction 1 protein could be produced, it was separated from other proteins by absorption of a leaf extract on to a column of immobilized antibodies raised against Nicotiuncr tubacum Fraction 1 protein (7), or by precipitation of the protein by an antiserum followed by carboxymethylation of the antibody-protein complex prior to isoelectrofocusing (8). The use of carboxymethylated protein which requires a preliminary puri’ This study Belgium.
was supported
by a&ant
ofthe
fication of the enzyme has been proposed by Kung et al. (4) and is currently used by other investigators in similar studies (9- 11). In this paper a method is described whereby Fraction 1 protein subunits can be separated into their polypeptides without preliminary purification steps and with only the use of combined electrophoretic and electrofocusing systems. Danno (12) described a general method for isoelectrofocusing of proteins attempted directly on the proteins in SDS-polyacrylamide’ gel slices. We adapted this technique to the analysis of the subunit composition of Fraction 1 protein. We succeeded in resolving the polypeptide composition of Fraction 1 protein by separating the enzyme first on a native polyacrylamide gel prior to isoelectrofocusing. MATERIALS
Leaf homogenates of a few Lycopersicon species were used to perform the experiments. Seeds were kindly provided by Professor C. M. Rick, University of California, Davis.
NFWO. L Abbreviation 317
used:
SDS.
sodium
dodecyl
sulfate.
0003.2697/80/1X0317-04$02.00/O CopyrIght 0 1980 by Academic Press. Inc. 411 nghtq of rrpruduction in any form rrwrvrd.
318
CAMMAERTSANDJACOBS
METHODS I, /soelectric
focusing
performed
on
Extraction. Fully expanded leaves were ground in a mortar in sample buffer (0.0625 M Tris-HCI, pH 6.8, 18% glycerol, 0.5% 2-mercaptoethanol) using 1 ml buffer for 1 g leaf. The slurry was centrifuged for 25 min at 35,500g. 0.08 g Sephadex G-25 was added to 1 ml of supernatant, and the remaining supernatant was submitted to electrophoresis. Polyucrylamide gel Plt~ctrophoresis. A discontinuous buffer system with an Ornstein (13) and Davis (14) stacking system was used as described by Laemmli (15) but modified as SDS was omitted. The separation gel contained 4.5% acrylamide and a final buffer concentration of 0.375 M Tris-HCI (pH 8.8). The stacking gel of 3% acrylamide (I cm length) contained 0.125 M Tris-HCI (pH 6.8). The electrode buffer (pH 8.3) contained 0.025 M Tris and 0.192 M glycine. The slab gel system described by Studier (16) was used and gels of a 18 cm length and 1.5 mm thickness were prepared. A maximum of 30 ~1 of sample was applied per sample well of 5 mm width. Gels were run until the bromophenol blue marker reached the bottom of the gel. The running time was about 4 h. Fraction 1 protein was visualized by staining for total protein for 2 to 5 min in a solution containing 0.1% Coomassie brilliant blue R-250, 50% methanol, and 10%’ acetic acid. and destaining in a solution of 5% methanol and 10% acetic acid for a few minutes. The position of Fraction 1 protein can easily be determined on account of its slow migration and its high staining intensity. Prepuration of t/w Fraction I protein gel slices. The Fraction 1 protein band was cut from the gel and shaken at room temperature for IO to 15 min in a small amount
of buffer A (2% LKB Ampholine, pH 5-8. 8 M urea. 5% sucrose) according to the method described by Danno (12). The gel slices can be stored in the freezer (-20°C) overnight or can be used immediately. Isoelectric, focusing in polytrcrylumidc ael. Isoelectric focusing was performed in thin layers of acrylamide (LKB 2117 Multiphor system). Gels containing 2% Ampholine (two parts pH 5-8. one part. pH 3.5-10). 8 M urea, 4.85% acrylamide, and 0.15% methylenebisacrylamide were prepared. Polymerization was obtained by addition of 2.7 x IO-“% riboflavine. The anodic strip was wet with I M phosphoric acid, the cathodic strip with a I M NaOH solution. The gel slices which were cut out of the polyacrylamide gel were transferred into holes made into the isoelectric focusing gel near the cathodic strip. Isoelectric focusing was performed at a constant power of 25 W for a thin-layer gel of 12 x 24 cm and at a final voltage of 1350 V. To resolve the small subunit polypeptides, a run of maximum 1.5 h was satisfactory and 3 h was needed for obtaining the pattern of the large subunit polypeptides. Staining. The slab gel was stained according to the method of Reisner (17). Ampholine and urea were removed from the slab gel by soaking the gel in 3.5% perchloric acid for at least 1 h. Gels were stained in a solution of 0.04% Coomassie brilliant blue G-250 in 3.5% perchloric acid at 37°C. 2. Isot>lectric, jhcusing performed on protein
An alternative method consists in bringing the Fraction 1 protein gel slices cut out of a polyacrylamide gel into the wells of a 12% SDS-polyacrylamide gel. The SDS-polyacrylamide gel electrophoresis was carried out according to the method described by Laemmli ( 15). Fraction 1protein can thus be senarated L ~~~ into its laree and small subunits.
ELECTROPHORETIC
TECHNIQUES
FOR
DETERMINING
SUBUNIT
319
POLYPEPTIDES
i
FIG. I. Separation of Fraction 4.5% polyacrylamide gel using Lywprrsic.on species. Fraction as one broad band.
1 protein on a native leaf homogenates of I protein is present
After electrophoresis, the gel is stained according to the method previously described under 1. and the large and small subunits are cut out of the gel. The gel slices are treated in the same manner as in method 1. and isoelectric focusing is carried out using the same conditions as described before.
FIG. 2. (Al Isoelectric focusing of Fraction I protein species performed on of different f.ycoprr.\icon Fraction I protein gel slices obtained by separation on a native polyacrylamide gel and showing the large subunit polypeptides (LSl. The different Lycoprrsicort species have large subunits whose cluster of three polypeptides have the same isoelectric points. (Bl Isoelectric focusing of Fraction I protein large subunit gel slices obtained by separation on SDS-polyacrylamide gel electrophoresis.
the results obtained for different Lycospecies will be published elsewhere. To be sure we had avoided possible contamination of Fraction I protein by other
pcrsicon
B
A
RESULTS AND DISCUSSION
The method described consists in separating Fraction 1 protein on a native polyacrylamide gel in which the enzyme can very easily be identified owing to its high molecular weight and relative amount (Fig. 1). The Fraction 1 protein bands were cut out of the gel and the gel slices were applied on an isoelectric focusing gel in the presence of 8 M urea without an elution step. In order to obtain a complete liberation of the protein out of the gel slices the use of polyacrylamide gels with low acrylamide concentration is necessary. We obtained a clear-cut separation in three large subunit polypeptides and in two to three small subunit polypeptides species depending on which Lycoprrsicon was analyzed (Figs. 2A. 3A). The report of
1
-
2
-
1 .a=n-
2 --
FIN,. 3. (A) Isoelectric focusing of Fraction I protein of two I,vco~micon species performed directly on Fraction I protein gel slices obtained by separation on native polyacrylamide gel and showing the small subunit polypeptides (SS). tB) Isoelectric focusing of Fraction I protein small subunit gel slices obtained by separation on SDS-polyacrylamide gel and performed on the same two Lyc’qwrsicon species as in (Al. (I) L. ~scrrlrnrum. (2) L. prruvinnunz.
320
CAMMAERTS
FIG. 4. Isoelectric focusing of Fraction I protein of L~coprrsicon and Nicotiuna species performed on Fraction 1 protein gel slices obtained by separation on native polyacrylamide gel and showing the small subunit polypeptides (SS), (1) L. ~sculentum. (2) N. tcthacum. (3) N. glutinosu. (4) N. ,&ucu. (5) N. svlvestris.
proteins migrating at the same position on the gel, we first separated the enzyme in its subunits on a SDS-polyacrylamide gel. Isoelectric focusing was carried out directly on the subunits present in SDS-polyacrylamide gel slices and the same polypeptide composition was obtained as when using native gel as shown in Figs. 2B and 3B. A very small amount of tissue is required to isolate Fraction 1 protein and to resolve the enzyme in its polypeptides. This characteristic makes the described method very suitable in studies of polymorphism as one leaf is enough to perform the analysis while the plant can be preserved for other purposes. The techniques used until now require different treatments of the enzyme prior to isoelectrofocusing. One of these treatments consists in carboxymethylating the isolated enzyme before resolving it on an isoelectric focusing gel to avoid the appearance of a high number of peptide bands due to oxidation of thiol groups exposed during dissociation of the subunits in 8 M urea (4). We have applied our method to resolve Fraction 1 protein of leaf extracts of Nicatianu species, without preIiminary
AND
JACOBS
carboxymethylation. Figure 4 shows a separation in small subunit polypeptides without the occurrence of many additional bands. The number of polypeptides observed for the different Nicotianu species is in agreement with results already published (18,19). species the application For Lycvpersicon of our method leads to a polypeptide composition comparable with those obtained from other techniques (20,21) but which require purification of leaf extracts and treatment of the enzyme prior to isoelectrofocusing and which are thus more laborious and time consuming. REFERENCES 1. Kawashima. N., and Wildman, S. (1970) Annu. Rev. Plant Physiol. 21, 325. 2. Kawashima, N., and Wildman. S. (1972) Biochim. Biophys. Acru 262, 42. 3. Chan. P.. and Wildman, S. (1972) Biochim. Biophy.P. Ac,tcr 277, 677. 4. Kung. S., Sakano. K.. and Wildman, S. ( 1974) Biochim. Bir&ys. A~,ta 365, 138. 5. Chen, K.. Kung, S.. Gray, J., and Wildman. S. (1976) Plant Sci. Lett. 7. 429. 6. Chan, K., Sakano. K., Singh, S., and Wildman. S. (1972) Science 176, 1145. 7. Gray, J.. and Wildman, S. (1976) Plunt Sci. Lett. 6, 91. 8. Uchimiya. H., Chen. K.. and Wildman, S. (1979) Plant Sci. Lett. 14, 387. 9. Gatenby. A.. and Cocking. E. (1977) Plant Sci. Lert. 10, 97. IO. Gatenby. A., and Cocking, E. (1978) Plant Sci. LrIt. 12, 177. 11. Steer, M., and Kernoghan. D. (1977) Biochem. Genet. 15, 273. 12. Danno, G. (1977) Ana/. Bi~chcm. 83, 189. 13. Omstein, L. (1964) Ann. N.Y. Acad. Sci. 121, 321. 14. Davis, B. ( 1964) Ann. N. Y. Acad. Sci. 121, 404. 15. Laemmli, U. (1970) Noture (London) 227, 680. 16. Studier. F. (1973) J. Mol. Biof. 79, 237. 17: Reisner. A.. Nemes, P., and Bucholtz. C. (1975) Anal. Biochem. 64, 509. 18. Sakano. K., Kung, S., and Wildman, S. (1974) Mol. Gen. Genet. 130, 91. 19. Gray, J., Kung, S., Wildman, S., and Sheen, S. (1974) Nature (London) 252. 226. 20. Gatenby, A., and Cocking, E. (1978) Plant Sci. Lett. 13, 171. 21. Uchimiya, H.. Chen. K., and Wildman, S. (1979) Biochrm. Genrt. 17, 333.
109, 3 17-320
BIOCHEMISTRY
(1980)
A Simple Electrophoretic Polypeptide Composition D. Lahorutorium
Procedure for the Determination of the of the Subunits of Fraction 1 Protein’
CAMMAERTS
AND
vow Plantmgenetica. B-1640 St.-Genesius-Rodeo Received
April
M. Vrijr
JACOBS Univrrsiteit Belgium
Bnrssel.
29. 1980
A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 M urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.
Fraction 1 protein (ribulosebiphosphate carboxylase EC 4.1.1.39), the major soluble protein in leaves of higher plants, is composed of large and small subunits (1). The large subunit has been shown to be chloroplast coded and maternally inherited, whereas the small subunit is coded by nuclear DNA (2.3). The polypeptide composition of the subunits of Fraction 1 protein has been determined by isoelectric focusing in 8 M urea using samples of purified carboxymethylated Fraction 1 protein (4,5). Fraction 1 protein was purified either by crystallization (6) or by salt fractionation and column chromatography (5). In the case where no crystalline Fraction 1 protein could be produced, it was separated from other proteins by absorption of a leaf extract on to a column of immobilized antibodies raised against Nicotiuncr tubacum Fraction 1 protein (7), or by precipitation of the protein by an antiserum followed by carboxymethylation of the antibody-protein complex prior to isoelectrofocusing (8). The use of carboxymethylated protein which requires a preliminary puri’ This study Belgium.
was supported
by a&ant
ofthe
fication of the enzyme has been proposed by Kung et al. (4) and is currently used by other investigators in similar studies (9- 11). In this paper a method is described whereby Fraction 1 protein subunits can be separated into their polypeptides without preliminary purification steps and with only the use of combined electrophoretic and electrofocusing systems. Danno (12) described a general method for isoelectrofocusing of proteins attempted directly on the proteins in SDS-polyacrylamide’ gel slices. We adapted this technique to the analysis of the subunit composition of Fraction 1 protein. We succeeded in resolving the polypeptide composition of Fraction 1 protein by separating the enzyme first on a native polyacrylamide gel prior to isoelectrofocusing. MATERIALS
Leaf homogenates of a few Lycopersicon species were used to perform the experiments. Seeds were kindly provided by Professor C. M. Rick, University of California, Davis.
NFWO. L Abbreviation 317
used:
SDS.
sodium
dodecyl
sulfate.
0003.2697/80/1X0317-04$02.00/O CopyrIght 0 1980 by Academic Press. Inc. 411 nghtq of rrpruduction in any form rrwrvrd.
318
CAMMAERTSANDJACOBS
METHODS I, /soelectric
focusing
performed
on
Extraction. Fully expanded leaves were ground in a mortar in sample buffer (0.0625 M Tris-HCI, pH 6.8, 18% glycerol, 0.5% 2-mercaptoethanol) using 1 ml buffer for 1 g leaf. The slurry was centrifuged for 25 min at 35,500g. 0.08 g Sephadex G-25 was added to 1 ml of supernatant, and the remaining supernatant was submitted to electrophoresis. Polyucrylamide gel Plt~ctrophoresis. A discontinuous buffer system with an Ornstein (13) and Davis (14) stacking system was used as described by Laemmli (15) but modified as SDS was omitted. The separation gel contained 4.5% acrylamide and a final buffer concentration of 0.375 M Tris-HCI (pH 8.8). The stacking gel of 3% acrylamide (I cm length) contained 0.125 M Tris-HCI (pH 6.8). The electrode buffer (pH 8.3) contained 0.025 M Tris and 0.192 M glycine. The slab gel system described by Studier (16) was used and gels of a 18 cm length and 1.5 mm thickness were prepared. A maximum of 30 ~1 of sample was applied per sample well of 5 mm width. Gels were run until the bromophenol blue marker reached the bottom of the gel. The running time was about 4 h. Fraction 1 protein was visualized by staining for total protein for 2 to 5 min in a solution containing 0.1% Coomassie brilliant blue R-250, 50% methanol, and 10%’ acetic acid. and destaining in a solution of 5% methanol and 10% acetic acid for a few minutes. The position of Fraction 1 protein can easily be determined on account of its slow migration and its high staining intensity. Prepuration of t/w Fraction I protein gel slices. The Fraction 1 protein band was cut from the gel and shaken at room temperature for IO to 15 min in a small amount
of buffer A (2% LKB Ampholine, pH 5-8. 8 M urea. 5% sucrose) according to the method described by Danno (12). The gel slices can be stored in the freezer (-20°C) overnight or can be used immediately. Isoelectric, focusing in polytrcrylumidc ael. Isoelectric focusing was performed in thin layers of acrylamide (LKB 2117 Multiphor system). Gels containing 2% Ampholine (two parts pH 5-8. one part. pH 3.5-10). 8 M urea, 4.85% acrylamide, and 0.15% methylenebisacrylamide were prepared. Polymerization was obtained by addition of 2.7 x IO-“% riboflavine. The anodic strip was wet with I M phosphoric acid, the cathodic strip with a I M NaOH solution. The gel slices which were cut out of the polyacrylamide gel were transferred into holes made into the isoelectric focusing gel near the cathodic strip. Isoelectric focusing was performed at a constant power of 25 W for a thin-layer gel of 12 x 24 cm and at a final voltage of 1350 V. To resolve the small subunit polypeptides, a run of maximum 1.5 h was satisfactory and 3 h was needed for obtaining the pattern of the large subunit polypeptides. Staining. The slab gel was stained according to the method of Reisner (17). Ampholine and urea were removed from the slab gel by soaking the gel in 3.5% perchloric acid for at least 1 h. Gels were stained in a solution of 0.04% Coomassie brilliant blue G-250 in 3.5% perchloric acid at 37°C. 2. Isot>lectric, jhcusing performed on protein
An alternative method consists in bringing the Fraction 1 protein gel slices cut out of a polyacrylamide gel into the wells of a 12% SDS-polyacrylamide gel. The SDS-polyacrylamide gel electrophoresis was carried out according to the method described by Laemmli ( 15). Fraction 1protein can thus be senarated L ~~~ into its laree and small subunits.
ELECTROPHORETIC
TECHNIQUES
FOR
DETERMINING
SUBUNIT
319
POLYPEPTIDES
i
FIG. I. Separation of Fraction 4.5% polyacrylamide gel using Lywprrsic.on species. Fraction as one broad band.
1 protein on a native leaf homogenates of I protein is present
After electrophoresis, the gel is stained according to the method previously described under 1. and the large and small subunits are cut out of the gel. The gel slices are treated in the same manner as in method 1. and isoelectric focusing is carried out using the same conditions as described before.
FIG. 2. (Al Isoelectric focusing of Fraction I protein species performed on of different f.ycoprr.\icon Fraction I protein gel slices obtained by separation on a native polyacrylamide gel and showing the large subunit polypeptides (LSl. The different Lycoprrsicort species have large subunits whose cluster of three polypeptides have the same isoelectric points. (Bl Isoelectric focusing of Fraction I protein large subunit gel slices obtained by separation on SDS-polyacrylamide gel electrophoresis.
the results obtained for different Lycospecies will be published elsewhere. To be sure we had avoided possible contamination of Fraction I protein by other
pcrsicon
B
A
RESULTS AND DISCUSSION
The method described consists in separating Fraction 1 protein on a native polyacrylamide gel in which the enzyme can very easily be identified owing to its high molecular weight and relative amount (Fig. 1). The Fraction 1 protein bands were cut out of the gel and the gel slices were applied on an isoelectric focusing gel in the presence of 8 M urea without an elution step. In order to obtain a complete liberation of the protein out of the gel slices the use of polyacrylamide gels with low acrylamide concentration is necessary. We obtained a clear-cut separation in three large subunit polypeptides and in two to three small subunit polypeptides species depending on which Lycoprrsicon was analyzed (Figs. 2A. 3A). The report of
1
-
2
-
1 .a=n-
2 --
FIN,. 3. (A) Isoelectric focusing of Fraction I protein of two I,vco~micon species performed directly on Fraction I protein gel slices obtained by separation on native polyacrylamide gel and showing the small subunit polypeptides (SS). tB) Isoelectric focusing of Fraction I protein small subunit gel slices obtained by separation on SDS-polyacrylamide gel and performed on the same two Lyc’qwrsicon species as in (Al. (I) L. ~scrrlrnrum. (2) L. prruvinnunz.
320
CAMMAERTS
FIG. 4. Isoelectric focusing of Fraction I protein of L~coprrsicon and Nicotiuna species performed on Fraction 1 protein gel slices obtained by separation on native polyacrylamide gel and showing the small subunit polypeptides (SS), (1) L. ~sculentum. (2) N. tcthacum. (3) N. glutinosu. (4) N. ,&ucu. (5) N. svlvestris.
proteins migrating at the same position on the gel, we first separated the enzyme in its subunits on a SDS-polyacrylamide gel. Isoelectric focusing was carried out directly on the subunits present in SDS-polyacrylamide gel slices and the same polypeptide composition was obtained as when using native gel as shown in Figs. 2B and 3B. A very small amount of tissue is required to isolate Fraction 1 protein and to resolve the enzyme in its polypeptides. This characteristic makes the described method very suitable in studies of polymorphism as one leaf is enough to perform the analysis while the plant can be preserved for other purposes. The techniques used until now require different treatments of the enzyme prior to isoelectrofocusing. One of these treatments consists in carboxymethylating the isolated enzyme before resolving it on an isoelectric focusing gel to avoid the appearance of a high number of peptide bands due to oxidation of thiol groups exposed during dissociation of the subunits in 8 M urea (4). We have applied our method to resolve Fraction 1 protein of leaf extracts of Nicatianu species, without preIiminary
AND
JACOBS
carboxymethylation. Figure 4 shows a separation in small subunit polypeptides without the occurrence of many additional bands. The number of polypeptides observed for the different Nicotianu species is in agreement with results already published (18,19). species the application For Lycvpersicon of our method leads to a polypeptide composition comparable with those obtained from other techniques (20,21) but which require purification of leaf extracts and treatment of the enzyme prior to isoelectrofocusing and which are thus more laborious and time consuming. REFERENCES 1. Kawashima. N., and Wildman, S. (1970) Annu. Rev. Plant Physiol. 21, 325. 2. Kawashima, N., and Wildman. S. (1972) Biochim. Biophys. Acru 262, 42. 3. Chan. P.. and Wildman, S. (1972) Biochim. Biophy.P. Ac,tcr 277, 677. 4. Kung. S., Sakano. K.. and Wildman, S. ( 1974) Biochim. Bir&ys. A~,ta 365, 138. 5. Chen, K.. Kung, S.. Gray, J., and Wildman. S. (1976) Plant Sci. Lett. 7. 429. 6. Chan, K., Sakano. K., Singh, S., and Wildman. S. (1972) Science 176, 1145. 7. Gray, J.. and Wildman, S. (1976) Plunt Sci. Lett. 6, 91. 8. Uchimiya. H., Chen. K.. and Wildman, S. (1979) Plant Sci. Lett. 14, 387. 9. Gatenby. A.. and Cocking. E. (1977) Plant Sci. Lert. 10, 97. IO. Gatenby. A., and Cocking, E. (1978) Plant Sci. LrIt. 12, 177. 11. Steer, M., and Kernoghan. D. (1977) Biochem. Genet. 15, 273. 12. Danno, G. (1977) Ana/. Bi~chcm. 83, 189. 13. Omstein, L. (1964) Ann. N.Y. Acad. Sci. 121, 321. 14. Davis, B. ( 1964) Ann. N. Y. Acad. Sci. 121, 404. 15. Laemmli, U. (1970) Noture (London) 227, 680. 16. Studier. F. (1973) J. Mol. Biof. 79, 237. 17: Reisner. A.. Nemes, P., and Bucholtz. C. (1975) Anal. Biochem. 64, 509. 18. Sakano. K., Kung, S., and Wildman, S. (1974) Mol. Gen. Genet. 130, 91. 19. Gray, J., Kung, S., Wildman, S., and Sheen, S. (1974) Nature (London) 252. 226. 20. Gatenby, A., and Cocking, E. (1978) Plant Sci. Lett. 13, 171. 21. Uchimiya, H.. Chen. K., and Wildman, S. (1979) Biochrm. Genrt. 17, 333.