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Proflavin-induced mutations in the l -arabinose operon of Escherichia coli
J. Mol. Biol. (1969) 46, 17-23
Pro&win-induced
Mutations in the wirabinose of Escherichia coli
Operon
II. Enzyme Analyses of the Mutants SUZANNE SESNOWITZ-HORN? AND EDWARD A. ADELBERQ
Department of Mhmbiology, Yale University New Haven, Conn. 06510, U.S.A. (Received 4 August 1969) The enzymic activities of mutant gene products, as well 88 the enzymic activities of products from adjacent genes, were assayed in strains harboring proflavininduced frame-shift mutations in the L-arabinose @a) operon. Such mutations in araB were found to depress strongly the expression of the adjacent gene, araA. Frame-shift mutations in the regulatory gene, araC, caused slight but significant depression of araA activity ; possible mechanisms for these pleiotropic effects are discussed. Frame-shift mutations in araB totally abolished the activity of L-ribulokinase, the product of the araB gene. Revertants, produced by second-site frame-shift mutations within the same gene, were found to have levels of L-ribulokinase lower than wild type.
1. Introduction The genes (araA, aruB, araD) which determine the structures of the enzymes for L-arabinose utilization, have been shown by Englesberg and his collaborators to constitute an operon, under positive control by the immediately adjacent regulatory gene, araC (Englesberg, Irr, Power & Lee, 1965). In the preceding paper (SesnowitzHorn & Adelberg, 1969), we have described the isolation of a series of proflavininduced frame-shift mutations in the araB and araC genes. The present paper reports the effects of these mutations on certain enzyme activities. We have measured three sets of enzyme activities. First, we have measured the level of the araB gene product, L-ribulokinase, in haploid strains carrying frame-shift mutations in the araB gene itself, as well as in proflavin-induced revertants of these mutants. Second, we have measured the level of the araA gene product, L-arabinoae isomerase, in merodiploid strains of the general type F-A-B+/A+B-, where Brepresents a frame-shift mutation in araB. The episomic element, F-A-B+, was introduced into these strains for the following reason. It has been observed (Cribbs & Englesberg, 1964) that various mutations in araB cause widely different inducible levels of isomerase (the araA gene product). Some mutations depress the isomerase level, whereas others greatly increase it (“hyperinducibility”). Katz t Englesberg (1968) have found that the hyperinducibility effect is abolished in diploids carrying a normal aruB+ allele in addition to the aruB- mutation. Thus, the construction of i Present address: Department of Developmental Biology and Center, Albert Einstein Medical College, Bronx, New York, U.S.A. 2
17
18
S. SESNOWITZ-HORN
AND
E. A. ADELBERG
the F-A-B+/A+Bmerodiploids permitted us to determine whether frame-shift mutations in the B gene affect the expression of the adjacent araA gene in the absence of the hyperinducibility phenomenon. Third, we have measured the level of the araA gene product, n-arabinose isomerase, in merodiploids of the general type F-A-C+/A+C-, where C- represents a frame-shift mutation in araC. In this case, the episome carrying the araC+ allele was introduced in order to activate the ara operon, which is totally unexpressed in an araC- haploid (Englesberg et al., 1965). The use of A -C+/A +C- diploids permitted us to determine whether frame-shift mutations in araC exert a pleiotropic effect on the expression of araA.
2. Materials and Methods (a) Media Assay medizcm. This medium, a modification of that described by Sheppard & Englesberg (1967), consists of a miners1 base (07% KzHPOI, 0.3% KHzPO,, 0.1% (NH&SO, and 0.01 yo MgSO,) plua l*O% Casamino acids, 0.5% yeast extract, with or without 0.4% L-arabinose (Y.C.A. or Y.C.A.A.). All other media used, as well as the various genetic procedures and strains, are described in the preceding con-mu&&ion (Sesnowitz-Horn & Adelberg, 1969). Merodiploids were constructed by the transfer of the episome F-aruC + -araB + -araAS, from a strain of Eecherichia co&i B supplied by Dr Ellis Englesberg. In order to introduce the episome from E. coli B into E. co.% K12, it was necessary first to provide the latter strains with the restriction and modification genes of E. coli B, by transduction with phage Pl (Boyer, 1964). The merodiploids were found to be unstable, producing new combinations of araA, araB and araC alleles by recombination between the episome and chromosome at a rate high enough to affect the results of the enzyme assays significantly. To overcome this problem, the haploid frame-shift mutants were first made thymineless by the method of Stacey & Simson (1965), and then co-transduced to recA-thy+ from 8 suitable donor, using phage Pl. F-merogenotes were then introduced into the recAstrains, where they were found to produce completely stable heterozygotes. (b) Culture
methods
Cells to be used in enzymic studies were prepared by growing the various strains overnight in 30 ml. of Y.C.A. medium at 37°C with aeration. In the morning, 2-1. Fernbach flasks containing 300 ml. of Y.C.A.A. medium were inoculated with these cells to give approximately 2.0 x lo* cells/ml. The cultures were incubated at 37°C with vigorous aeration for several hours until a cell density of 6.0 to 8.0 x lo* cells/ml. was reached as estimated from reading in the Klett-Summerson calorimeter. (c) Preparation
of cell-free
extracts
Cultures, grown as previously described, were harvested during logarithmic growth. A sample (0.1 ml.) of each was tested for purity by streaking on eosin methylene blue L-arabinose agar. The cells were sedimented by centrifugation at 9000 rev./minfor 10 min in a Sorvall RC-2 refrigerated centrifuge at 0°C. The supernatant fraction was discarded and the pellet washed in approximately 100 ml. of 1.0 x 10m3 M-glutathione, and 1.0 M-glycylglycine buffer (pH 7.6) to give a final volume of 4.0 ml. This suspension was sonicated for 2 min at 1.4 A and 118 v in an MSE sonicator. The sonicated material w&s centrifuged at 15,000 rev./min at 0°C for 30 min to remove cell debris. The supernatant fraction was collected. (d) Enzyme
assays
L-Arabinose isomeruse. The activity of this enzyme was measured by a modification of the method described by Cribbs & Englesberg (1964). The reaction mixture consisted of: 126 pmoles glycylglycine buffer (pH 7.6), 2.4 pmoles MnCl,, 150 pmoles L-arabinose, and cell extract, in a final volume of 1.0 ml. The extract was added to small Wasserman tubes containing the reaction mixture in an ice bath. Each tube was then placed in a 30°C stationary water bath and O*l-ml. samples were removed at 5-min intervals for 20 min
PROFLAVIN-INDUCED
MUTATIONS.
II
19
into tubes containing 0.9 ml. of 0.1 M-HCl. Assays for L-ribulose were performed directly on these samples using the cystein+carbazole test (Dische & Borenfreund, 1951). The color produced was determined exactly 20 min after the addition of reagents, using a Klett-Summerson calorimeter with the green filter (22.5 Klett units = 4.45 X lo4 pmoles n-ribulose). Specific activity was defined as the number of micromoles of n-ribulose formed/hr/mg of protein. Controls (reaction mixtures containing no cell extracts) were treated in exactly the same manner. L-Ribulokinase. The assay procedure was a modification of that described by Lee & Englesberg (1963). The reaction contains: 42 qoles Tris (pH 7.4) ; 4 pmoles glutathione; 8 pmoles ATP ; and 2 pmoles L2 pmoles EDTA; 20 moles MgCls ; 20 pmoles NaF; [1-‘*C]ribulose in a total volume of O-1 ml. Samples of the cell extract were added to small Wasserman tubes containing the reaction mixture at O”C, which were then incubated in a 37°C stationary water bath for either 6 or 15 min, depending upon the level of activity. The reaction was stopped by the addition of absolute ethanol (0.8 ml. for 6-min incubations and 1.6 ml. for 15-min reactions) at 0°C. The phosphorylated L-ribulose was precipitated by the addition of 1.0 M-barium acetate (0.1 ml. or O-2 ml., depending on activity). After incubating for 15 min at O”C, the precipitates were collected on membrane filters ((Millipore, HA 0.45 mm), washed 6 times with l-O-ml. portions of cold 80% ethanol, and placed in counting vials. Toluene POP-di-POP solution (Packard Instrument Co.) was added to the air-dried filters. The radioactivity was determined with a NuclearChicago liquid scintillation counter. A sample containing no cell extract was always included as a control. Specific enzyme activity was defined as the number of micromoles of L-ribulose phosphorylated/hr/mg protein.
(e) Protein determination Protein was estimated by the method described by Lowry, Rosebrough, Farr & Randall, (1951). Crystalline bovine serum albumin was used as a standard. Samples of each extract were measured in duplicate.
3. Results (a) L-Rib&k&use
activity in eraB frame-shift mutants and their revertants
The L-ribulokinase activities of some araB frame-shift mutants and their revertants are shown in Table 1. There was no detectable L-ribulokinrtse activity in any of the frame-shift mutants; the revertants possessed L-ribulokinase levels lower than that of wild type. Certain of these revertants were previously shown to be “pseudowild types,” e.g. they carry two frame-shift mutations within the araB gene (SesnowitzHorn & Adelberg, 1969). (b) Isomerase levels in F-A-B
+/A +B - merodiploids
The results of this set of measurements s,re presented in Table 2. As a control on the of measuring isomerese levels in such diploids, a missense mutant (induced by 2-aminopurine) has been included. The induced level (specific activity) of isomersse in an A +B + haploid is 13.6 ho.78 units of enzyme per mg of protein, In the A+ B- haploid missense mutant, the induced isomerase level is 24.5. In the P-A-B+/A+Bdiploid carrying the same araB missense mutation, the isomerase level is 12.7 ; thus the introduction of the F-A-B+ episome is completely effective in eliminating the hyperinducibility phenomenon. Given these controls, it can be seen that six of the nine araB frame-shift mutations have caused a significant depression of the isomerase activity, which ranges from 11y0 (araB25) to 60% (uraBI6) of the control level. validity
20
S. SESNOWITZ-HORN
AND
E. A.
ADELBERG
TABLE 1 Induced L-ribulokinase levels in araB frame-shift mutants and in pseudowild-type araB revertants (double mutants)
Mutest
r,-Ribulokinaset Experimentt Average 1 2
~&lea
Primary mutints araBl6 araBI araB19 araB&O araB21 araB22 amB23 araB24 araB25 Double araBl6, araBl8, araB22, araB16, araBl6, araBl6,
mutents araB20 araB31 araB32 araB33 araB34 araB35
:
to.1 to-1
: 3.4 2.1 2.1 4.9 6.0 6.9
4.0 2.8 2.3
3.7 2.5 2.2 4.9 6.0 5.9
t Enzyme wtivity expressed aa pmoles L-ribulose phosphorylated/hr/mg activity = 10.0 (average of five experiments). 1 Each assay wa8 performed on a freshly prepared extmct.
protein.
Wild type
TABLET Indeed
levels of L-arabinose isomerme (product of the araA gene) in merodiploids (F-A-B+/A+B-)t
Proflavin-induced mutation
araB
araBl6 araBl8 araBl9 araB20 araB21 araB22 araB23 araB24 araB25 Missense mutant Wild type (A +B + heploid)
1 7.6 9.9 4.6 4.2 3.7 9.3 3.7 9.5 1.8 12.4
L-Arebinose isomerase$ Experiments 4 2 3 6.3 11.3 6.1 2.1 3.7 11.4 3.8 8.5 1.2 13-o
6.7 9.9 6.7 2.6
12.6
9.9
t Enzyme aotivity expressed aa qoles of L-ribulose formed/hr/mg $ Ewh assay was performed on & freshly prepared extract. 8 Averege of 10 determinations.
protein.
Average
6.3 10.9 5.1 2.9 3.7 10.2 3.8 9.0 1.5 12.7 13.6 f 0.78$
PROFLAVIN-INDUCED
MUTATIONS.
21
II.
(c) Isomeraae levels in F-A-C +/A+ C - merodiploids The results of this set of measurements are presented in Table 3. All six of the araC frame-shift mutations caused a slight depression in isomerase activity, which ranges from 55 to 85% of the wild-type haploid level.
3
TABLE
Induced L-arabinose isomerase levels in merodiploids (F-C +A-/C- A+ )t
Proflavin-induced mutation
araC 1
araCl7 araC27 araC28 araC29 araC3O Wild type (A + B + haploid)
7.1 13.0 8.3 10.3 9.9
L-Arabinose Experiments 2 10.9 9.9 6.4 10.0 5.5
isomerase$ 3
10.1
t Each assay was performed on a freshly prepared extract. $ Enzyme activity is expressed as pmoles of n-ribulose formed/hr/mg 8 Average of 10 determinations.
(d) Relation of pleiotropic
Average
9.0 11.6 7.4 10.2 8.5 136 f 0.788
of protein.
effects to map positions of the mutations
Figure 1 presents the isomerase levels (araA activity) associated with frame-shift mutations at different sites in the araB and araC genes. No correlation is observed between map position and the strength of the pleiotropic effect. B .-----lea
mix
19 :i
spec act of i-oroblnose ,somerase
23 24 5.1 3.7 102
25 29
29
6.3 109 15
E FIG. 1. Pleiotropic effects of proflavin-induced mutations in araB and araC: effect on expression of araA. Specific activity of L-arabinose isomerase is expressed as pmoles of L-ribulose formed/hr/mg of protein (from Tables 2 and 3). The map of the n-arabinose genes is reproduced from Sheppard & Englesberg (1967). The horizontal bars below the map indicate the regions in which are located the mutations whose numbers are listed underneath.
4. Discussion (a)
L-Ribulokinuse activity in araB frame-shift mutants and their revertants
The absence of detectable L-ribulokinase activity in the araB mutants is compatible with the conclusion, based on genetic evidence (&snow&z-Horn & Adelberg, 1969), that these mutants carry frame-shift mutations in aruB. Since a frameshift
22
S. SESNOWITZ-HORN
AND
E.
A. ADELBERG
mutation must alter every codon in the gene distal to the mutant site, an enzyme with partial activity would only be conceivable if the mutation had occurred in one of the last few codons of the gene. The finding that the revertants possess an L-ribulokinase with a specific activity lower than that of wild type is consistent with the genetic evidence, also presented in the preceding paper, that these revertants carry two frame-shift mutations which cancel the effect of each other except for a short sequence of altered codons between the mutant sites. (b) Isomerase levels in A-B+/A+Bmerodiploids Six of the nine strains carrying frame-shift mutations in araB were found to have significantly depressed levels of the adjacent (araA) gene product, L-arabinose isomerase. In the most extreme case, the isomerase activity was only 11% of that seen in a control diploid strain carrying a missense mutation in aruB, or in the haploid wild-type strain. The most likely explanation for the pleiotropic effect of the frame-shift mutations is that they are polar: i.e. they not only abolish the activity of the product of the gene in which they are located, but also reduce the level of expression of those genes in the same operon which are distal to the mutant locus. Such polarity of pleiotropic effects has been demonstrated for nonsense mutations, which not only cause premature polypeptide chain termination during translation, but also lower the probability that the ribosome will initiate a new polypeptide chain when it reaches the next initiation site on the polygenic messenger RNA molecule (Martin, Whitfield, Berkowitz & Voll, 1966). Frame-shift mutations can be predicted, on simple statistical grounds, to produce an average of one nonsense codon for every 64 nucleotides that the ribosome must travel beyond the mutant site. Thus, the average frame-shift mutation will generate several nonsense codons in the gene in which it occurs, and as a result should exhibit a polarity effect (Whitfield, Martin & Ames, 1966). It might also be predicted that a frame-shift mutation which did not generate a nonsense codon (i.e. one occurring at the very end of the gene) would be absolutely polar, as a result of the frame-shift carrying through into the next gene; mutants which would permit a test of this prediction, however, have not yet been found. Frame-shift mutations in the histidine operon exhibit polarity (Martin, Silbert, Smith & Whitfield, 1966) ; the effects of the araB frame-shift mutations on araA expression are probably due to the same mechanism. Proof of translational polarity, however, requires the demonstration that the mutation in question affects distal (away from the operator) but not proximal genes of the operon. The gene order in the present instance is araC-aruB-araA-araD (Englesberg et al., 1965). Evidence has been presented that araC is not part of the operon by Sheppard & Englesberg (1967), who have also discussed the evidence for the existence of an “initiator” site between araB and araC, which suggests the direction of reading of the operon is B-A-D, and thus that araA is distal to aruB. We have not, however, been able to measure effects of araB mutations on araC gene expression, so that the polarity of the araB frameshift mutations must remain as a tentative explanation of their pleiotropic effects. (c) Isomeruse levels in F-A-C +/A+ C - merodiploids Since the frame-shift mutations in araC reduced the isomerase levels by very small amounts (15 to 45% reduction), it is difficult to interpret their effects. If, as discussed
PROFLAVIN-INDUCED
MUTATIONS.
II
23
by Sheppard & Englesberg (1967), araC is not part of the L-arabinose operon, the pleiotropic effects of the araC frame-shift mutations could reflect alterations in the efficacy of the positive control mechanism. (d) Relation of pleiotropic
effect to wmp position
Since we have attributed the effects on isomerase, of at least the araB frame-shift mutations, to translational polarity, we have asked whether there is any relationship of the degree of polarity to map position. In the lac operon (Newton, Beckwith, Zipser & Brenner, 1965) and in the tryptophan operon (Yanofsky & Ito, 1966), a correlation exists between the location of a nonsense mutation and the degree of polarity: the closer the mutation to the proximal end of the gene, the stronger the effect. As indicated in Pigure 1, however, no such “polarity gradient” is found in the case of ara frame-shift mutations. We thank Dr Ellis Englesberg for his co-operation the enzyme assay procedures in his laboratory.
and for the opportunity
to learn
REFERENCES Boyer, H. (1964). J. Boot. 88, 1652. Cribbs, R. & Englesberg, E. (1964). Genetics, 49, 95. Dische, Z. & Borenfreund, E. (1951). J. Biol. Chem. 192, 583. Englesberg, E., Irr, J., Power, J. & Lee, N. (1965). J. Bact. 90, 946. Katz, L. & Englesberg, E. (1968). Bact. Proc. p. 50. Lee, N. & Englesberg, E. (1963). Proc. Nat. Acad. Sci., Wash. 50, 696. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, J. (1951). J. Biol. Chem. 193,266. Martin, R. G., Silbert, D. F., Smith, D. W. E. & Whitfield, H. J., Jr. (1966). J. Mol. Biol. 21, 357. Martin, R. G., Whitfield, H. J., Jr. Berkowitz, D. B. & Voll, M. J. (1966). Cold Spr. Had. Symp. Qumt. BioZ. 31, 215. Newton, W., Beckwith, J. R., Zipser, D. & Brenner, S. (1965). J. Mol. BioZ. 14, 290. Sesnowitz-Horn, S. & Adelberg, E. A. (1969). J. Mol. BioZ. 46, 1. Sheppard, D. & Englesberg, E. (1967). J. MOE. BioZ. 25, 443. Stacey, K. A. & Simson, E. (1965). J. Bad. 90, 554. Whitfield, H. J., Jr., Martin, R. & Ames, B. (1966). J. Mol. BioZ. 21, 335. Yanofsky, C. & Ito, J. (1966). J. Mol. Biol. 21, 313.
Pro&win-induced
Mutations in the wirabinose of Escherichia coli
Operon
II. Enzyme Analyses of the Mutants SUZANNE SESNOWITZ-HORN? AND EDWARD A. ADELBERQ
Department of Mhmbiology, Yale University New Haven, Conn. 06510, U.S.A. (Received 4 August 1969) The enzymic activities of mutant gene products, as well 88 the enzymic activities of products from adjacent genes, were assayed in strains harboring proflavininduced frame-shift mutations in the L-arabinose @a) operon. Such mutations in araB were found to depress strongly the expression of the adjacent gene, araA. Frame-shift mutations in the regulatory gene, araC, caused slight but significant depression of araA activity ; possible mechanisms for these pleiotropic effects are discussed. Frame-shift mutations in araB totally abolished the activity of L-ribulokinase, the product of the araB gene. Revertants, produced by second-site frame-shift mutations within the same gene, were found to have levels of L-ribulokinase lower than wild type.
1. Introduction The genes (araA, aruB, araD) which determine the structures of the enzymes for L-arabinose utilization, have been shown by Englesberg and his collaborators to constitute an operon, under positive control by the immediately adjacent regulatory gene, araC (Englesberg, Irr, Power & Lee, 1965). In the preceding paper (SesnowitzHorn & Adelberg, 1969), we have described the isolation of a series of proflavininduced frame-shift mutations in the araB and araC genes. The present paper reports the effects of these mutations on certain enzyme activities. We have measured three sets of enzyme activities. First, we have measured the level of the araB gene product, L-ribulokinase, in haploid strains carrying frame-shift mutations in the araB gene itself, as well as in proflavin-induced revertants of these mutants. Second, we have measured the level of the araA gene product, L-arabinoae isomerase, in merodiploid strains of the general type F-A-B+/A+B-, where Brepresents a frame-shift mutation in araB. The episomic element, F-A-B+, was introduced into these strains for the following reason. It has been observed (Cribbs & Englesberg, 1964) that various mutations in araB cause widely different inducible levels of isomerase (the araA gene product). Some mutations depress the isomerase level, whereas others greatly increase it (“hyperinducibility”). Katz t Englesberg (1968) have found that the hyperinducibility effect is abolished in diploids carrying a normal aruB+ allele in addition to the aruB- mutation. Thus, the construction of i Present address: Department of Developmental Biology and Center, Albert Einstein Medical College, Bronx, New York, U.S.A. 2
17
18
S. SESNOWITZ-HORN
AND
E. A. ADELBERG
the F-A-B+/A+Bmerodiploids permitted us to determine whether frame-shift mutations in the B gene affect the expression of the adjacent araA gene in the absence of the hyperinducibility phenomenon. Third, we have measured the level of the araA gene product, n-arabinose isomerase, in merodiploids of the general type F-A-C+/A+C-, where C- represents a frame-shift mutation in araC. In this case, the episome carrying the araC+ allele was introduced in order to activate the ara operon, which is totally unexpressed in an araC- haploid (Englesberg et al., 1965). The use of A -C+/A +C- diploids permitted us to determine whether frame-shift mutations in araC exert a pleiotropic effect on the expression of araA.
2. Materials and Methods (a) Media Assay medizcm. This medium, a modification of that described by Sheppard & Englesberg (1967), consists of a miners1 base (07% KzHPOI, 0.3% KHzPO,, 0.1% (NH&SO, and 0.01 yo MgSO,) plua l*O% Casamino acids, 0.5% yeast extract, with or without 0.4% L-arabinose (Y.C.A. or Y.C.A.A.). All other media used, as well as the various genetic procedures and strains, are described in the preceding con-mu&&ion (Sesnowitz-Horn & Adelberg, 1969). Merodiploids were constructed by the transfer of the episome F-aruC + -araB + -araAS, from a strain of Eecherichia co&i B supplied by Dr Ellis Englesberg. In order to introduce the episome from E. coli B into E. co.% K12, it was necessary first to provide the latter strains with the restriction and modification genes of E. coli B, by transduction with phage Pl (Boyer, 1964). The merodiploids were found to be unstable, producing new combinations of araA, araB and araC alleles by recombination between the episome and chromosome at a rate high enough to affect the results of the enzyme assays significantly. To overcome this problem, the haploid frame-shift mutants were first made thymineless by the method of Stacey & Simson (1965), and then co-transduced to recA-thy+ from 8 suitable donor, using phage Pl. F-merogenotes were then introduced into the recAstrains, where they were found to produce completely stable heterozygotes. (b) Culture
methods
Cells to be used in enzymic studies were prepared by growing the various strains overnight in 30 ml. of Y.C.A. medium at 37°C with aeration. In the morning, 2-1. Fernbach flasks containing 300 ml. of Y.C.A.A. medium were inoculated with these cells to give approximately 2.0 x lo* cells/ml. The cultures were incubated at 37°C with vigorous aeration for several hours until a cell density of 6.0 to 8.0 x lo* cells/ml. was reached as estimated from reading in the Klett-Summerson calorimeter. (c) Preparation
of cell-free
extracts
Cultures, grown as previously described, were harvested during logarithmic growth. A sample (0.1 ml.) of each was tested for purity by streaking on eosin methylene blue L-arabinose agar. The cells were sedimented by centrifugation at 9000 rev./minfor 10 min in a Sorvall RC-2 refrigerated centrifuge at 0°C. The supernatant fraction was discarded and the pellet washed in approximately 100 ml. of 1.0 x 10m3 M-glutathione, and 1.0 M-glycylglycine buffer (pH 7.6) to give a final volume of 4.0 ml. This suspension was sonicated for 2 min at 1.4 A and 118 v in an MSE sonicator. The sonicated material w&s centrifuged at 15,000 rev./min at 0°C for 30 min to remove cell debris. The supernatant fraction was collected. (d) Enzyme
assays
L-Arabinose isomeruse. The activity of this enzyme was measured by a modification of the method described by Cribbs & Englesberg (1964). The reaction mixture consisted of: 126 pmoles glycylglycine buffer (pH 7.6), 2.4 pmoles MnCl,, 150 pmoles L-arabinose, and cell extract, in a final volume of 1.0 ml. The extract was added to small Wasserman tubes containing the reaction mixture in an ice bath. Each tube was then placed in a 30°C stationary water bath and O*l-ml. samples were removed at 5-min intervals for 20 min
PROFLAVIN-INDUCED
MUTATIONS.
II
19
into tubes containing 0.9 ml. of 0.1 M-HCl. Assays for L-ribulose were performed directly on these samples using the cystein+carbazole test (Dische & Borenfreund, 1951). The color produced was determined exactly 20 min after the addition of reagents, using a Klett-Summerson calorimeter with the green filter (22.5 Klett units = 4.45 X lo4 pmoles n-ribulose). Specific activity was defined as the number of micromoles of n-ribulose formed/hr/mg of protein. Controls (reaction mixtures containing no cell extracts) were treated in exactly the same manner. L-Ribulokinase. The assay procedure was a modification of that described by Lee & Englesberg (1963). The reaction contains: 42 qoles Tris (pH 7.4) ; 4 pmoles glutathione; 8 pmoles ATP ; and 2 pmoles L2 pmoles EDTA; 20 moles MgCls ; 20 pmoles NaF; [1-‘*C]ribulose in a total volume of O-1 ml. Samples of the cell extract were added to small Wasserman tubes containing the reaction mixture at O”C, which were then incubated in a 37°C stationary water bath for either 6 or 15 min, depending upon the level of activity. The reaction was stopped by the addition of absolute ethanol (0.8 ml. for 6-min incubations and 1.6 ml. for 15-min reactions) at 0°C. The phosphorylated L-ribulose was precipitated by the addition of 1.0 M-barium acetate (0.1 ml. or O-2 ml., depending on activity). After incubating for 15 min at O”C, the precipitates were collected on membrane filters ((Millipore, HA 0.45 mm), washed 6 times with l-O-ml. portions of cold 80% ethanol, and placed in counting vials. Toluene POP-di-POP solution (Packard Instrument Co.) was added to the air-dried filters. The radioactivity was determined with a NuclearChicago liquid scintillation counter. A sample containing no cell extract was always included as a control. Specific enzyme activity was defined as the number of micromoles of L-ribulose phosphorylated/hr/mg protein.
(e) Protein determination Protein was estimated by the method described by Lowry, Rosebrough, Farr & Randall, (1951). Crystalline bovine serum albumin was used as a standard. Samples of each extract were measured in duplicate.
3. Results (a) L-Rib&k&use
activity in eraB frame-shift mutants and their revertants
The L-ribulokinase activities of some araB frame-shift mutants and their revertants are shown in Table 1. There was no detectable L-ribulokinrtse activity in any of the frame-shift mutants; the revertants possessed L-ribulokinase levels lower than that of wild type. Certain of these revertants were previously shown to be “pseudowild types,” e.g. they carry two frame-shift mutations within the araB gene (SesnowitzHorn & Adelberg, 1969). (b) Isomerase levels in F-A-B
+/A +B - merodiploids
The results of this set of measurements s,re presented in Table 2. As a control on the of measuring isomerese levels in such diploids, a missense mutant (induced by 2-aminopurine) has been included. The induced level (specific activity) of isomersse in an A +B + haploid is 13.6 ho.78 units of enzyme per mg of protein, In the A+ B- haploid missense mutant, the induced isomerase level is 24.5. In the P-A-B+/A+Bdiploid carrying the same araB missense mutation, the isomerase level is 12.7 ; thus the introduction of the F-A-B+ episome is completely effective in eliminating the hyperinducibility phenomenon. Given these controls, it can be seen that six of the nine araB frame-shift mutations have caused a significant depression of the isomerase activity, which ranges from 11y0 (araB25) to 60% (uraBI6) of the control level. validity
20
S. SESNOWITZ-HORN
AND
E. A.
ADELBERG
TABLE 1 Induced L-ribulokinase levels in araB frame-shift mutants and in pseudowild-type araB revertants (double mutants)
Mutest
r,-Ribulokinaset Experimentt Average 1 2
~&lea
Primary mutints araBl6 araBI araB19 araB&O araB21 araB22 amB23 araB24 araB25 Double araBl6, araBl8, araB22, araB16, araBl6, araBl6,
mutents araB20 araB31 araB32 araB33 araB34 araB35
:
to.1 to-1
: 3.4 2.1 2.1 4.9 6.0 6.9
4.0 2.8 2.3
3.7 2.5 2.2 4.9 6.0 5.9
t Enzyme wtivity expressed aa pmoles L-ribulose phosphorylated/hr/mg activity = 10.0 (average of five experiments). 1 Each assay wa8 performed on a freshly prepared extmct.
protein.
Wild type
TABLET Indeed
levels of L-arabinose isomerme (product of the araA gene) in merodiploids (F-A-B+/A+B-)t
Proflavin-induced mutation
araB
araBl6 araBl8 araBl9 araB20 araB21 araB22 araB23 araB24 araB25 Missense mutant Wild type (A +B + heploid)
1 7.6 9.9 4.6 4.2 3.7 9.3 3.7 9.5 1.8 12.4
L-Arebinose isomerase$ Experiments 4 2 3 6.3 11.3 6.1 2.1 3.7 11.4 3.8 8.5 1.2 13-o
6.7 9.9 6.7 2.6
12.6
9.9
t Enzyme aotivity expressed aa qoles of L-ribulose formed/hr/mg $ Ewh assay was performed on & freshly prepared extract. 8 Averege of 10 determinations.
protein.
Average
6.3 10.9 5.1 2.9 3.7 10.2 3.8 9.0 1.5 12.7 13.6 f 0.78$
PROFLAVIN-INDUCED
MUTATIONS.
21
II.
(c) Isomeraae levels in F-A-C +/A+ C - merodiploids The results of this set of measurements are presented in Table 3. All six of the araC frame-shift mutations caused a slight depression in isomerase activity, which ranges from 55 to 85% of the wild-type haploid level.
3
TABLE
Induced L-arabinose isomerase levels in merodiploids (F-C +A-/C- A+ )t
Proflavin-induced mutation
araC 1
araCl7 araC27 araC28 araC29 araC3O Wild type (A + B + haploid)
7.1 13.0 8.3 10.3 9.9
L-Arabinose Experiments 2 10.9 9.9 6.4 10.0 5.5
isomerase$ 3
10.1
t Each assay was performed on a freshly prepared extract. $ Enzyme activity is expressed as pmoles of n-ribulose formed/hr/mg 8 Average of 10 determinations.
(d) Relation of pleiotropic
Average
9.0 11.6 7.4 10.2 8.5 136 f 0.788
of protein.
effects to map positions of the mutations
Figure 1 presents the isomerase levels (araA activity) associated with frame-shift mutations at different sites in the araB and araC genes. No correlation is observed between map position and the strength of the pleiotropic effect. B .-----lea
mix
19 :i
spec act of i-oroblnose ,somerase
23 24 5.1 3.7 102
25 29
29
6.3 109 15
E FIG. 1. Pleiotropic effects of proflavin-induced mutations in araB and araC: effect on expression of araA. Specific activity of L-arabinose isomerase is expressed as pmoles of L-ribulose formed/hr/mg of protein (from Tables 2 and 3). The map of the n-arabinose genes is reproduced from Sheppard & Englesberg (1967). The horizontal bars below the map indicate the regions in which are located the mutations whose numbers are listed underneath.
4. Discussion (a)
L-Ribulokinuse activity in araB frame-shift mutants and their revertants
The absence of detectable L-ribulokinase activity in the araB mutants is compatible with the conclusion, based on genetic evidence (&snow&z-Horn & Adelberg, 1969), that these mutants carry frame-shift mutations in aruB. Since a frameshift
22
S. SESNOWITZ-HORN
AND
E.
A. ADELBERG
mutation must alter every codon in the gene distal to the mutant site, an enzyme with partial activity would only be conceivable if the mutation had occurred in one of the last few codons of the gene. The finding that the revertants possess an L-ribulokinase with a specific activity lower than that of wild type is consistent with the genetic evidence, also presented in the preceding paper, that these revertants carry two frame-shift mutations which cancel the effect of each other except for a short sequence of altered codons between the mutant sites. (b) Isomerase levels in A-B+/A+Bmerodiploids Six of the nine strains carrying frame-shift mutations in araB were found to have significantly depressed levels of the adjacent (araA) gene product, L-arabinose isomerase. In the most extreme case, the isomerase activity was only 11% of that seen in a control diploid strain carrying a missense mutation in aruB, or in the haploid wild-type strain. The most likely explanation for the pleiotropic effect of the frame-shift mutations is that they are polar: i.e. they not only abolish the activity of the product of the gene in which they are located, but also reduce the level of expression of those genes in the same operon which are distal to the mutant locus. Such polarity of pleiotropic effects has been demonstrated for nonsense mutations, which not only cause premature polypeptide chain termination during translation, but also lower the probability that the ribosome will initiate a new polypeptide chain when it reaches the next initiation site on the polygenic messenger RNA molecule (Martin, Whitfield, Berkowitz & Voll, 1966). Frame-shift mutations can be predicted, on simple statistical grounds, to produce an average of one nonsense codon for every 64 nucleotides that the ribosome must travel beyond the mutant site. Thus, the average frame-shift mutation will generate several nonsense codons in the gene in which it occurs, and as a result should exhibit a polarity effect (Whitfield, Martin & Ames, 1966). It might also be predicted that a frame-shift mutation which did not generate a nonsense codon (i.e. one occurring at the very end of the gene) would be absolutely polar, as a result of the frame-shift carrying through into the next gene; mutants which would permit a test of this prediction, however, have not yet been found. Frame-shift mutations in the histidine operon exhibit polarity (Martin, Silbert, Smith & Whitfield, 1966) ; the effects of the araB frame-shift mutations on araA expression are probably due to the same mechanism. Proof of translational polarity, however, requires the demonstration that the mutation in question affects distal (away from the operator) but not proximal genes of the operon. The gene order in the present instance is araC-aruB-araA-araD (Englesberg et al., 1965). Evidence has been presented that araC is not part of the operon by Sheppard & Englesberg (1967), who have also discussed the evidence for the existence of an “initiator” site between araB and araC, which suggests the direction of reading of the operon is B-A-D, and thus that araA is distal to aruB. We have not, however, been able to measure effects of araB mutations on araC gene expression, so that the polarity of the araB frameshift mutations must remain as a tentative explanation of their pleiotropic effects. (c) Isomeruse levels in F-A-C +/A+ C - merodiploids Since the frame-shift mutations in araC reduced the isomerase levels by very small amounts (15 to 45% reduction), it is difficult to interpret their effects. If, as discussed
PROFLAVIN-INDUCED
MUTATIONS.
II
23
by Sheppard & Englesberg (1967), araC is not part of the L-arabinose operon, the pleiotropic effects of the araC frame-shift mutations could reflect alterations in the efficacy of the positive control mechanism. (d) Relation of pleiotropic
effect to wmp position
Since we have attributed the effects on isomerase, of at least the araB frame-shift mutations, to translational polarity, we have asked whether there is any relationship of the degree of polarity to map position. In the lac operon (Newton, Beckwith, Zipser & Brenner, 1965) and in the tryptophan operon (Yanofsky & Ito, 1966), a correlation exists between the location of a nonsense mutation and the degree of polarity: the closer the mutation to the proximal end of the gene, the stronger the effect. As indicated in Pigure 1, however, no such “polarity gradient” is found in the case of ara frame-shift mutations. We thank Dr Ellis Englesberg for his co-operation the enzyme assay procedures in his laboratory.
and for the opportunity
to learn
REFERENCES Boyer, H. (1964). J. Boot. 88, 1652. Cribbs, R. & Englesberg, E. (1964). Genetics, 49, 95. Dische, Z. & Borenfreund, E. (1951). J. Biol. Chem. 192, 583. Englesberg, E., Irr, J., Power, J. & Lee, N. (1965). J. Bact. 90, 946. Katz, L. & Englesberg, E. (1968). Bact. Proc. p. 50. Lee, N. & Englesberg, E. (1963). Proc. Nat. Acad. Sci., Wash. 50, 696. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, J. (1951). J. Biol. Chem. 193,266. Martin, R. G., Silbert, D. F., Smith, D. W. E. & Whitfield, H. J., Jr. (1966). J. Mol. Biol. 21, 357. Martin, R. G., Whitfield, H. J., Jr. Berkowitz, D. B. & Voll, M. J. (1966). Cold Spr. Had. Symp. Qumt. BioZ. 31, 215. Newton, W., Beckwith, J. R., Zipser, D. & Brenner, S. (1965). J. Mol. BioZ. 14, 290. Sesnowitz-Horn, S. & Adelberg, E. A. (1969). J. Mol. BioZ. 46, 1. Sheppard, D. & Englesberg, E. (1967). J. MOE. BioZ. 25, 443. Stacey, K. A. & Simson, E. (1965). J. Bad. 90, 554. Whitfield, H. J., Jr., Martin, R. & Ames, B. (1966). J. Mol. BioZ. 21, 335. Yanofsky, C. & Ito, J. (1966). J. Mol. Biol. 21, 313.