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Genetic evidence on the nature of the repressor for alkaline phosphatase in E. coli
J. Mol. Biol. (1963) 6, 433-438
Genetic Evidence on the Nature of the Repressor for Alkaline Phosphatase in E. coli ALAN GAREN AND SUZANNE GAREN
Division of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. (Received 14 January 1963) Certain constitutive mutations in the two regulator genes for alkaline phosphatase respond to an external suppressor. This result suggests that the products of the regulator genes, and therefore the repressor for alkaline phosphatase, are protein molecules.
1. Introduction The role of repressor substances in the regulation of the rate of protein synthesis has been described for several enzymes of E. coli (Jacob & Monod, 1961; Echols, Garen, Garen & Torriani, 1961; Cohen & Jacob, 1959; Gorini, Gundersen & Burger, 1961; Maas, 1961; Buttin, 1961; Yarmolinsky, Jordan , Wiesmeyer, Kalckar & Sundararjan, 1961). A general characteristic of repressors is their ability to act through the cytoplasm to prevent specifically the synthesis of certain enzymes. Formation of a particular repressor is genetically controlled by one or more regulator genes. Much of our current knowledge about the action of repressors has been obtained by genetic analysis of constitutive mutants in which repressor formation has been blocked by mutations in regulator genes. As yet there has been no direct study of the chemical nature of repressor substances. The only chemical information bearing on this point is that the repressor for ,B-galactosidase can be formed in the presence of chloramphenicol or 5-methyltryptophan, which are inhibitors of protein synthesis (Pardee & Prestidge, 1959). By this criterion the repressor for ,B-galactosidase would appear not to be a protein. In the present report we have used a genetic test for determining whether the repressor for alkaline phosphatase is a protein. The test is based on the action of an external suppressor in E. coli that suppresses certain mutations in the structural cistron for alkaline phosphatase (Garen & Siddiqi, 1962) and also certain mutations in the rII cistrons of phage T4 (Benzer & Champe, 1962). In the absence of the suppressor, these mutations in the phosphatase structural cistron prevent the synthesis of the enzyme. The presence of the suppressor restores the ability to synthesize the enzyme, thereby reversing the mutant phenotype. A mechanism for the action of this suppressor has been proposed (Benzer & Champe, 1962 ; Garen & Siddiqi, 1962) which involves one of the steps in protein synthesis. If this mechanism is to be effective in suppressing a mutation, the mutation must be located in a cistron that specifies a protein. Accordingly, we have tested the effect of the suppressor on various constitutive mutations in the R1 and R2 regulator genes, which control the formation of a repressor for alkaline phosphatase (Echols et al., 1961). Some of the mutations 433
434
A. GAREN AND S. GAREN
in both genes showed a strong response to the suppressor. This result suggests that products of both genes are proteins. A similar test involving an external suppressor has been applied to mutations in the 91 regulator gene of phage A by Jacob, Sussman & Monod (1962). The 01 mutants are unable to lysogenize strain 112 of E. coli K12. It was found that the ability to lysogenize, and therefore presumably to produce a repressor, could be restored to certain 01 mutants when a suppressor strain of 112 was used as host. Since this suppressor also acts on mutations in various structural genes of phage ,\ and on bacterial mutations affecting the utilization of galactose and the synthesis of cysteine, it was concluded that the product of the 01 regulator gene is a protein molecule.
2. Materials and Methods (a) Genetic nomenclature of bacterial strains
Hfr: Male (genetic donor) strain. F-: Female (genetic recipient) strain. P+: The phosphatase-positive strain containing the standard structural cistron for alkaline phosphatase. su+: A strain which can suppress certain phosphatase-negative mutat.ions in the structural cistron (Garen & Siddiqi, 1962). su-: A strain without the active su+ suppressor. RI+ and R2+: The standard types for the two phosphatase regulator genes. An RI+R2+P+ strain produces a large amount of alkaline phosphatase in low phosphate medium and is repressed in high phosphate medium (Garen & Levinthal, 1960; Echols et al., 1961). RI-: A phosphatase-constitutive (non-repressible) mutant resulting from a mutation in the RI regulator gene. R2-: A phosphatase-constitutive mutant resulting from a mutation in the R2 regulator gene. T+ and T-: Threonine-independent (T+) or dependent (T-) strains. L+ and L-: Leucine-independent (L+) or dependent (L-) strains. S8 and sr: Streptomycin-sensitive (S") or resistant (sr) strains (to a level of 0·1 mg streptomycin 1m I. ). (b) Suppressor character of bacterial strains The standard Hfr (KI0) which was used for all experiments is a non-suppressing strain (su-). The standard F- is a suppressing strain (su+). For certain experiments requiring a non-suppressing F-, a strain was produced by crossing the standard F- with the Hfr and selecting a recombinant F- having the eu»: character of the Hfr (Garen & Siddiqi, 1962). (c) Isolation of phosphatase-constitutive mutants
The constitutive mutants were isolated in Hfr strain KI0 by the procedure of Torriani & Rothman (1961). (d) Classification of phosphatase-constitutive mutations as Rl or R2
Phosphatase-constitutive mutations can occur in either of two regulator genes, RI or R2 (Echols et al., 1961). The location of such a constitutive R- mutation can be determined from the result of the following cross: Hfr R- P+T+L+S8 S U - x F- R+P+T-L-BTsu-. If the R- mutation is located in the RI gene, approximately 60% of the selected T+ L+sr recombinants from the cross will be R-, while if the location is in the R2 gene, approxi- . mately 3% of the selected recombinants will be R- (Echols et al., 1961). Since in this cross both the Hfr and F- parents have the identical su: marker, an R- mutation is
NATURE OF ALKALINE PHOSPHATASE REPRESSOR
435
not subject to suppression. Therefore, the R phenotype of the recombinants can be scored without the ambiguity that might otherwise arise because of the suppressibility of an R - mutation. (e) Preparation of phosphatase-constitutive strains
The test for suppression of a phosphatase-constitutive R- mutation requires two strains, one with the suppressor (su+) and another without (su-), each carrying the same constitutive mutation. The su» constibut.ive strain was prepared by crossing an Hfr Rrsu»:P+T+L+S" with an F- Rrsu:"P+T- LrS" and isolating an R-P+T+ L+Srrecombinant. (For the procedure of this type of cross see Echols et al., 1961.) Since the Hfr transfers its su marker very late, and therefore inefficiently (Garen & Siddiqi, 1962), it has been assumed that the selected recombinant retains the eu» character of the F- parent. The su : constitutive strain was prepared in a similar way, by crossing the same constitutive Hfr with an F- R+su-T-L-S' and again isolating an R-P+T+L+sr recombinant. If a particular R- constitutive mutation is strongly suppressed in the su+ strain, the R-su+ recombinants that arise in the above cross with an su+ strain are phenotypically R+ (repressible) and therefore indistinguishable from the F- parental type. In this case, the su+ strain is prepared by crossing the Hfr mutant with an F- that has a nonsuppressible R- constitutive mutation (in the same R gene as the R- mutation in the Hfr) and isolating a recombinant that is phenotypically R+. (f) Assay for alkaline phosphatase enzymic activity Cultures were grown with aeration to a density of about 3 x 10s/ml. in a medium of the following composition: 1·2 x 10- 1 M-tris buffer, pH 7'5; 6·4 x 10-4 M-KH 2PO C ; 0·2% glucose; 8 x 10-2 M-NaCI; 2 x 10- 2 M-KCI; 2 x 10- 2 M-NHcCI; 3 x 10- 3 M-Na2SOc; 1 x 10-2 M-MgCI2; 2 x 10-4 M-CaCI2; 2 x 10- 6 M-FeCI 3 ; 0·01% Difco bacto-peptone, This concentration of orthophosphate is sufficient to repress alkaline phosphatase synthesis by the standard Rl +R2+ P+ strain. The cells were spun down and resuspended in an equal vol. of 0·1 M-tris buffer, pH 7·5. Absorbancy was read at 540 mfL in a 1 em cell. A few drops of toluene were added and the suspension was shaken at 37°C for 30 min. For enzymic assays, a 0·1 ml. sample of the toluenized suspension was added to 2·4 ml. of a solution of 1 M-tris buffer, pH 8,0, containing 4 mg/ml. of p-nitrophenylphosphate, and the rate of change of absorbancy at 410 mfL at 32°C was measured. The values for enzymic activities are normalized to a standard cell density of the culture.
3. Results The effect of the suppressor on Rl and R2 constitutive mutations is shown in Tables 1 and 2. The criterion for suppression is a loss of capacity to synthesize alkaline phosphatase constitutively (i.e. in high phosphate medium). It is evident that suppression does occur and that the extent varies markedly for different mutations. The most striking response is obtained with the Rl mutation C29 and the R2 mutation C84, both of which are totally suppressed. Other mutations are suppressed to lesser extents. It is important to establish whether the suppressor of constitutive mutations is identical to the suppressor that acts on mutations in the alkaline phosphatase structural cistron (Garen & Siddiqi, 1962). Although the fact that the same suppressor strains were used in studying both classes of mutations suggests that this is the case, there is still a possibility that different suppressors may be involved. The following experiment provides a critical test of this point. From the standard non-suppressing (su-) Hfr strain, a spontaneous su+ revertant, having suppressor activity for mutations in the phosphatase structural cistron, can be isolated (Garen & Siddiqi, 1962). The su+ marker from such a revertant was transferred, by mating, into the F- su-
436
A. GAREN AND S. GAREN
strain used in the present experiments. The resulting F- su+ recombinant was tested for its ability to suppress the Rl constitutive mutation 029 (see Table 1). The result again was complete suppression of the constitutive character when the 029 mutation TABLE
1
Response of various R1 constitutive mutants to the suppressor Rl mutation Standard strain itit» R2+)
02 05
06 07
09 026 027 028 029 030 032 033
036 074 080 082 083 085 088
094 095 099 0110 0111
Enzymic activity in high phosphate medium 8U - strain su + strain (suppressor absent) (suppressor present) 0·02
0-006
4·5 3·0 4·3 3·2 3·3 3·9 4·2 3·8 3·3 3·1 3·8 4·1 2·8 4·7 3·4 3·7 2·0 9·5 3·7 4·5 3·2 3·3 3·5 6·2
3·4 2·4 0·15 3·1 3·2 2·3 3·2 4·1 0·007 2·9
3·4 2·7 0·06 4·0 3·5 2·3 1·8 8·0 2·8 1·9 2·4 0·58 2·5 3·5
The sur: and su+ strains were prepared by transferring a constitutive mutation from the Hfr into the appropriate F-, as described under Materials and Methods. Several of the constitutive mutations (02, 05, 07 and 09) were previously shown to involve separate genetic sites (Echols et al., 1961). Each measurement was made in duplicate, and the results agreed to ± 10%.
was transferred to this F- su+ strain. This indicates that suppression of both constitutive mutations and mutations in the structural cistron is caused by the same suppressor.
4. Discussion The present finding that certain constitutive mutations in the Rl and R2 regulator genes for alkaline phosphatase respond to a particular suppressor provides genetic evidence that the product of each gene is a protein. Both regulator genes control essential steps in the formation of the repressor for alkaline phosphatase, and it
NATURE OF ALKALINE PHOSPHATASE REPRESSOR
437
appears that one of the genes specifies, at least in part, the repressor molecule (Echols et al., 1961; Garen & Echols, 1962a, b). These observations together suggest that the repressor for alkaline phosphatase is a protein molecule. T.ABLE
2
Response of various R2 constitutive mutants to the suppressor R2
mutation
Standard strain
Enzymic activity in high phosphate medium 8U- strain 8U+ strain (suppressor absent) (suppressor present) 0·02
0·006
(Rl+ R2+) 064
07·5 077 081 084
087 090 091 092 093 096
097 098 0101 0102 0105 0107 0108 0109 0112 0129 0130 0131 0132 0133
6·6 9·5 5·0 8·7 8·2 9·5 8·8 8·4 11·5 10·4 8·5 9·5 11·7 2·2 12·5 9·2 5·0 9·8 11·7 11·4 14·7 12·8 10·3 10·5 10·7
0·02 7·0 4·6 4·6 0·004 9·1 6·6 1·5 9·2 9·4 8·0 9·5 9·1 2·0 6·0 0·94 4·0 9·0 5·2 10·5 2·9 7·0 8·7 10·2 4·4
The procedures were the same as for Table 1.
This work was supported by grants from the National Science Foundation and National Institutes of Health, U.S. Public Health Service. We wish to acknowledge the technical assistance of Mrs. Rebecca Greer. REFERENCES Benzer, S. & Champe, S.P. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1114. Buttin, G. (1961). Cold Spr, Harb. Symp. Quant. Biol. 26, 213. Cohen, G. & Jacob, F. (1959). C.R. Acad. Sci., Paris, 248, 3490. Echols, H., Garen, A., Garen, S. & Torriani, A. (1961). J. Mol. Biol. 3, 425. Garen, A. & Echols, H. (1962a). J. Bact. 83, 297.
438
A. GAREN AND S. GAREN
Garen, A. & Echols, H. (l962b). Proc. Nat. Acad. Sci., WCUlh. 48, 1398. Garen, A. & Levinthal, C. (1960). Biochim. biophys. Acta, 38, 470. Garen, A. & Siddiqi, O. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1121. Gorini, L., Gundersen, W. & Burger, M. (1961). ColdSpr. Harb. Symp. Quant. Bioi. 26, 173. Jacob, F. & Monod, J. (1961). J. Mol. Biol. 3, 318. Jacob, F., Sussman, R. & Monod, J. (1962). C.R. Acad. s«, Paris, 254, 4214. Maas, W. K. (1961). Cold Spr, Harb. Symp. Quant. Biol. 26, 183. Pardee, A. B. & Prestidge, L. S. (1959). Biochim. biophys. Acta, 36, 545. Torriani, A. & Rothman, F. (1961). J. Bact. 81, 835. Yarmolinsky, M. B., Jordan, E., Wiesmeyer, R., Kalckar, R. M. & Sundararjan, T. A. (1961). Cold S'pr. Harb. Symp. Quant. Biol. 26, 217.
Genetic Evidence on the Nature of the Repressor for Alkaline Phosphatase in E. coli ALAN GAREN AND SUZANNE GAREN
Division of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. (Received 14 January 1963) Certain constitutive mutations in the two regulator genes for alkaline phosphatase respond to an external suppressor. This result suggests that the products of the regulator genes, and therefore the repressor for alkaline phosphatase, are protein molecules.
1. Introduction The role of repressor substances in the regulation of the rate of protein synthesis has been described for several enzymes of E. coli (Jacob & Monod, 1961; Echols, Garen, Garen & Torriani, 1961; Cohen & Jacob, 1959; Gorini, Gundersen & Burger, 1961; Maas, 1961; Buttin, 1961; Yarmolinsky, Jordan , Wiesmeyer, Kalckar & Sundararjan, 1961). A general characteristic of repressors is their ability to act through the cytoplasm to prevent specifically the synthesis of certain enzymes. Formation of a particular repressor is genetically controlled by one or more regulator genes. Much of our current knowledge about the action of repressors has been obtained by genetic analysis of constitutive mutants in which repressor formation has been blocked by mutations in regulator genes. As yet there has been no direct study of the chemical nature of repressor substances. The only chemical information bearing on this point is that the repressor for ,B-galactosidase can be formed in the presence of chloramphenicol or 5-methyltryptophan, which are inhibitors of protein synthesis (Pardee & Prestidge, 1959). By this criterion the repressor for ,B-galactosidase would appear not to be a protein. In the present report we have used a genetic test for determining whether the repressor for alkaline phosphatase is a protein. The test is based on the action of an external suppressor in E. coli that suppresses certain mutations in the structural cistron for alkaline phosphatase (Garen & Siddiqi, 1962) and also certain mutations in the rII cistrons of phage T4 (Benzer & Champe, 1962). In the absence of the suppressor, these mutations in the phosphatase structural cistron prevent the synthesis of the enzyme. The presence of the suppressor restores the ability to synthesize the enzyme, thereby reversing the mutant phenotype. A mechanism for the action of this suppressor has been proposed (Benzer & Champe, 1962 ; Garen & Siddiqi, 1962) which involves one of the steps in protein synthesis. If this mechanism is to be effective in suppressing a mutation, the mutation must be located in a cistron that specifies a protein. Accordingly, we have tested the effect of the suppressor on various constitutive mutations in the R1 and R2 regulator genes, which control the formation of a repressor for alkaline phosphatase (Echols et al., 1961). Some of the mutations 433
434
A. GAREN AND S. GAREN
in both genes showed a strong response to the suppressor. This result suggests that products of both genes are proteins. A similar test involving an external suppressor has been applied to mutations in the 91 regulator gene of phage A by Jacob, Sussman & Monod (1962). The 01 mutants are unable to lysogenize strain 112 of E. coli K12. It was found that the ability to lysogenize, and therefore presumably to produce a repressor, could be restored to certain 01 mutants when a suppressor strain of 112 was used as host. Since this suppressor also acts on mutations in various structural genes of phage ,\ and on bacterial mutations affecting the utilization of galactose and the synthesis of cysteine, it was concluded that the product of the 01 regulator gene is a protein molecule.
2. Materials and Methods (a) Genetic nomenclature of bacterial strains
Hfr: Male (genetic donor) strain. F-: Female (genetic recipient) strain. P+: The phosphatase-positive strain containing the standard structural cistron for alkaline phosphatase. su+: A strain which can suppress certain phosphatase-negative mutat.ions in the structural cistron (Garen & Siddiqi, 1962). su-: A strain without the active su+ suppressor. RI+ and R2+: The standard types for the two phosphatase regulator genes. An RI+R2+P+ strain produces a large amount of alkaline phosphatase in low phosphate medium and is repressed in high phosphate medium (Garen & Levinthal, 1960; Echols et al., 1961). RI-: A phosphatase-constitutive (non-repressible) mutant resulting from a mutation in the RI regulator gene. R2-: A phosphatase-constitutive mutant resulting from a mutation in the R2 regulator gene. T+ and T-: Threonine-independent (T+) or dependent (T-) strains. L+ and L-: Leucine-independent (L+) or dependent (L-) strains. S8 and sr: Streptomycin-sensitive (S") or resistant (sr) strains (to a level of 0·1 mg streptomycin 1m I. ). (b) Suppressor character of bacterial strains The standard Hfr (KI0) which was used for all experiments is a non-suppressing strain (su-). The standard F- is a suppressing strain (su+). For certain experiments requiring a non-suppressing F-, a strain was produced by crossing the standard F- with the Hfr and selecting a recombinant F- having the eu»: character of the Hfr (Garen & Siddiqi, 1962). (c) Isolation of phosphatase-constitutive mutants
The constitutive mutants were isolated in Hfr strain KI0 by the procedure of Torriani & Rothman (1961). (d) Classification of phosphatase-constitutive mutations as Rl or R2
Phosphatase-constitutive mutations can occur in either of two regulator genes, RI or R2 (Echols et al., 1961). The location of such a constitutive R- mutation can be determined from the result of the following cross: Hfr R- P+T+L+S8 S U - x F- R+P+T-L-BTsu-. If the R- mutation is located in the RI gene, approximately 60% of the selected T+ L+sr recombinants from the cross will be R-, while if the location is in the R2 gene, approxi- . mately 3% of the selected recombinants will be R- (Echols et al., 1961). Since in this cross both the Hfr and F- parents have the identical su: marker, an R- mutation is
NATURE OF ALKALINE PHOSPHATASE REPRESSOR
435
not subject to suppression. Therefore, the R phenotype of the recombinants can be scored without the ambiguity that might otherwise arise because of the suppressibility of an R - mutation. (e) Preparation of phosphatase-constitutive strains
The test for suppression of a phosphatase-constitutive R- mutation requires two strains, one with the suppressor (su+) and another without (su-), each carrying the same constitutive mutation. The su» constibut.ive strain was prepared by crossing an Hfr Rrsu»:P+T+L+S" with an F- Rrsu:"P+T- LrS" and isolating an R-P+T+ L+Srrecombinant. (For the procedure of this type of cross see Echols et al., 1961.) Since the Hfr transfers its su marker very late, and therefore inefficiently (Garen & Siddiqi, 1962), it has been assumed that the selected recombinant retains the eu» character of the F- parent. The su : constitutive strain was prepared in a similar way, by crossing the same constitutive Hfr with an F- R+su-T-L-S' and again isolating an R-P+T+L+sr recombinant. If a particular R- constitutive mutation is strongly suppressed in the su+ strain, the R-su+ recombinants that arise in the above cross with an su+ strain are phenotypically R+ (repressible) and therefore indistinguishable from the F- parental type. In this case, the su+ strain is prepared by crossing the Hfr mutant with an F- that has a nonsuppressible R- constitutive mutation (in the same R gene as the R- mutation in the Hfr) and isolating a recombinant that is phenotypically R+. (f) Assay for alkaline phosphatase enzymic activity Cultures were grown with aeration to a density of about 3 x 10s/ml. in a medium of the following composition: 1·2 x 10- 1 M-tris buffer, pH 7'5; 6·4 x 10-4 M-KH 2PO C ; 0·2% glucose; 8 x 10-2 M-NaCI; 2 x 10- 2 M-KCI; 2 x 10- 2 M-NHcCI; 3 x 10- 3 M-Na2SOc; 1 x 10-2 M-MgCI2; 2 x 10-4 M-CaCI2; 2 x 10- 6 M-FeCI 3 ; 0·01% Difco bacto-peptone, This concentration of orthophosphate is sufficient to repress alkaline phosphatase synthesis by the standard Rl +R2+ P+ strain. The cells were spun down and resuspended in an equal vol. of 0·1 M-tris buffer, pH 7·5. Absorbancy was read at 540 mfL in a 1 em cell. A few drops of toluene were added and the suspension was shaken at 37°C for 30 min. For enzymic assays, a 0·1 ml. sample of the toluenized suspension was added to 2·4 ml. of a solution of 1 M-tris buffer, pH 8,0, containing 4 mg/ml. of p-nitrophenylphosphate, and the rate of change of absorbancy at 410 mfL at 32°C was measured. The values for enzymic activities are normalized to a standard cell density of the culture.
3. Results The effect of the suppressor on Rl and R2 constitutive mutations is shown in Tables 1 and 2. The criterion for suppression is a loss of capacity to synthesize alkaline phosphatase constitutively (i.e. in high phosphate medium). It is evident that suppression does occur and that the extent varies markedly for different mutations. The most striking response is obtained with the Rl mutation C29 and the R2 mutation C84, both of which are totally suppressed. Other mutations are suppressed to lesser extents. It is important to establish whether the suppressor of constitutive mutations is identical to the suppressor that acts on mutations in the alkaline phosphatase structural cistron (Garen & Siddiqi, 1962). Although the fact that the same suppressor strains were used in studying both classes of mutations suggests that this is the case, there is still a possibility that different suppressors may be involved. The following experiment provides a critical test of this point. From the standard non-suppressing (su-) Hfr strain, a spontaneous su+ revertant, having suppressor activity for mutations in the phosphatase structural cistron, can be isolated (Garen & Siddiqi, 1962). The su+ marker from such a revertant was transferred, by mating, into the F- su-
436
A. GAREN AND S. GAREN
strain used in the present experiments. The resulting F- su+ recombinant was tested for its ability to suppress the Rl constitutive mutation 029 (see Table 1). The result again was complete suppression of the constitutive character when the 029 mutation TABLE
1
Response of various R1 constitutive mutants to the suppressor Rl mutation Standard strain itit» R2+)
02 05
06 07
09 026 027 028 029 030 032 033
036 074 080 082 083 085 088
094 095 099 0110 0111
Enzymic activity in high phosphate medium 8U - strain su + strain (suppressor absent) (suppressor present) 0·02
0-006
4·5 3·0 4·3 3·2 3·3 3·9 4·2 3·8 3·3 3·1 3·8 4·1 2·8 4·7 3·4 3·7 2·0 9·5 3·7 4·5 3·2 3·3 3·5 6·2
3·4 2·4 0·15 3·1 3·2 2·3 3·2 4·1 0·007 2·9
3·4 2·7 0·06 4·0 3·5 2·3 1·8 8·0 2·8 1·9 2·4 0·58 2·5 3·5
The sur: and su+ strains were prepared by transferring a constitutive mutation from the Hfr into the appropriate F-, as described under Materials and Methods. Several of the constitutive mutations (02, 05, 07 and 09) were previously shown to involve separate genetic sites (Echols et al., 1961). Each measurement was made in duplicate, and the results agreed to ± 10%.
was transferred to this F- su+ strain. This indicates that suppression of both constitutive mutations and mutations in the structural cistron is caused by the same suppressor.
4. Discussion The present finding that certain constitutive mutations in the Rl and R2 regulator genes for alkaline phosphatase respond to a particular suppressor provides genetic evidence that the product of each gene is a protein. Both regulator genes control essential steps in the formation of the repressor for alkaline phosphatase, and it
NATURE OF ALKALINE PHOSPHATASE REPRESSOR
437
appears that one of the genes specifies, at least in part, the repressor molecule (Echols et al., 1961; Garen & Echols, 1962a, b). These observations together suggest that the repressor for alkaline phosphatase is a protein molecule. T.ABLE
2
Response of various R2 constitutive mutants to the suppressor R2
mutation
Standard strain
Enzymic activity in high phosphate medium 8U- strain 8U+ strain (suppressor absent) (suppressor present) 0·02
0·006
(Rl+ R2+) 064
07·5 077 081 084
087 090 091 092 093 096
097 098 0101 0102 0105 0107 0108 0109 0112 0129 0130 0131 0132 0133
6·6 9·5 5·0 8·7 8·2 9·5 8·8 8·4 11·5 10·4 8·5 9·5 11·7 2·2 12·5 9·2 5·0 9·8 11·7 11·4 14·7 12·8 10·3 10·5 10·7
0·02 7·0 4·6 4·6 0·004 9·1 6·6 1·5 9·2 9·4 8·0 9·5 9·1 2·0 6·0 0·94 4·0 9·0 5·2 10·5 2·9 7·0 8·7 10·2 4·4
The procedures were the same as for Table 1.
This work was supported by grants from the National Science Foundation and National Institutes of Health, U.S. Public Health Service. We wish to acknowledge the technical assistance of Mrs. Rebecca Greer. REFERENCES Benzer, S. & Champe, S.P. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1114. Buttin, G. (1961). Cold Spr, Harb. Symp. Quant. Biol. 26, 213. Cohen, G. & Jacob, F. (1959). C.R. Acad. Sci., Paris, 248, 3490. Echols, H., Garen, A., Garen, S. & Torriani, A. (1961). J. Mol. Biol. 3, 425. Garen, A. & Echols, H. (1962a). J. Bact. 83, 297.
438
A. GAREN AND S. GAREN
Garen, A. & Echols, H. (l962b). Proc. Nat. Acad. Sci., WCUlh. 48, 1398. Garen, A. & Levinthal, C. (1960). Biochim. biophys. Acta, 38, 470. Garen, A. & Siddiqi, O. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1121. Gorini, L., Gundersen, W. & Burger, M. (1961). ColdSpr. Harb. Symp. Quant. Bioi. 26, 173. Jacob, F. & Monod, J. (1961). J. Mol. Biol. 3, 318. Jacob, F., Sussman, R. & Monod, J. (1962). C.R. Acad. s«, Paris, 254, 4214. Maas, W. K. (1961). Cold Spr, Harb. Symp. Quant. Biol. 26, 183. Pardee, A. B. & Prestidge, L. S. (1959). Biochim. biophys. Acta, 36, 545. Torriani, A. & Rothman, F. (1961). J. Bact. 81, 835. Yarmolinsky, M. B., Jordan, E., Wiesmeyer, R., Kalckar, R. M. & Sundararjan, T. A. (1961). Cold S'pr. Harb. Symp. Quant. Biol. 26, 217.