Eur. J. Biochem. 100, 165-173 (1979)
Abnormal Metabolism of Thymidine Nucleotides and Phosphorylation of Deoxycytidine in Escherichia coli C thy- ura- Mutant Tsutomu OHKAWA Department of Biochemistry, Kanazawa University School of Medicine (Received July 10, 1978 / J u n e 12, 1979)
The Escherichia coli C thy- ura- mutant (Thy A-35) shows a rate of DNA replication (or chain elongation) one-half that of the other thy- ura- mutant (Thy A-1 l), but RNA and protein syntheses in the Thy A-35 mutant show the same rates as those in the Thy A-1 1 mutant. The viable cell numbers in the Thy A-35 mutant exhibit a slightly slower rate than those in the Thy A-1 1 mutant. Thymidine and deoxycytidine triphosphate (dTTP and dCTP) pools in the Thy A-35 mutant increase 2 - 3-fold compared to those in the Thy A-1 1 mutant during exponential growth. As shown by Pritchard and Lond. B. Biol. Sci. (1974) 267, Zaritsky [J. Bacteriol. (1973) 114, 824-837; Philos. Trans. R. SOC. 303 - 3361 the reduced rate of DNA replication in the Thy A-35 mutant reduces the DNA concentration. Consequently, the differential rate of DNA synthesis in the Thy A-35 mutant is lower than that in other strains. Also, in the Thy A-35 mutant the rate of DNA replication in the culture with deoxycytidine added increases twofold compared to that in the culture without any addition, and this rate is the same as that of DNA replication in the Thy A-11 mutant. The thymidine triphosphate (dTTP) pool in the culture with deoxycytidine of the Thy A-35 mutant increases twofold compared to its value in the culture lacking deoxycytidine. It is concluded that DNA replications in both Thy A-1 1 and Thy A-35 mutants depend upon the intracellular dTTP pool. In vivo and in vitro the Thy A-35. mutant cells are able to phosphorylate deoxycytidine into deoxycytidine nucleotides, and have considerable deoxycytidine kinase or such enzyme which can activate the phosphorylation of deoxycytidine. For the purpose of studying the relationship between the thymidine triphosphate (dTTP) pool and deoxyribonucleic acid metabolism in E. coli, the thymine-requiring mutant is generally isolated. Recently it was reported that the replication time of the chromosome can be varied without a concomitant change in growth rate, simply by changing the concentration of thymine in the growth medium in thy- strains of E. coli [l - 31. In particular, when the thymine concentration in the growth medium of a thy- strain is reduced, the rate of DNA elongation is also reduced [I]. This effect reduces the average DNA concentration (the DNA : mass ratio). An increase Enzymes. Thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase (EC 2.4.2.4) ; thymidine kinase or ATP: thymine 5’-phosphotransferase (EC 2.7.1.21); cytidine (deoxycytidine) deaminase or cytidine (deoxycytidine) aminohydrolase (EC 3.5.4.5); cytidine (deoxycytidine) kinase or ATP:cytidine (deoxycytidine) 5’-phosphotransferase (EC 2.7.1.74); thymidylate synthase (EC 2.1.1.45).
in the growth rate of a thy+ culture has exactly the same effect on DNA concentration [3,4]. One mutant (Thy A-35) isolated here from E . coli C strain by aminopterin treatment shows a rate of DNA replication lower than that of other mutants under the same conditions. Therefore, it is considered that the average DNA concentration in the Thy A-35 mutant would be reduced more than that in other mutants. From this, it is surmised that the dTTP pool in the Thy A-35 mutant would be reduced more than that in other mutants, because the rate of DNA replication depends upon the size of the dTTP pool in thymutants of E. coli 15 T- and K12 T - [5,6]. Exogenous thymidine is readily incorporated into DNA in wild-type E. coli cells, but the incorporation soon ceases after a short time, as the enzyme thymidine phosphorylase degrades thymidine to thymine and deoxyribose I-phosphate [7]. Accordingly, mutants lacking this enzyme were able to incorporate thymidine for extended periods of time [8,9]. It is often assumed
166
that E. coli can incorporate deoxyribonucleosides other than thymidine as long as their concentrations in the medium are high enough, because both lactobacilli [lo] and mammalian systems [11] can utilize other deoxyribonucleosides for DNA synthesis. But the other deoxyribonucleosides have not been directly incorporated into DNA, as these compounds are rapidly catabolized in wild-type E. coli [12- 141. Karlstrom [15] has recently isolated the mutant (OK 441) that lacks four deoxyribonucleoside-catabolizing enzymes [16] to measure directly the incorporation of four deoxyribonucleosides. This mutant (OK 441) is unable to incorporate significant amounts of deoxycytidine, deoxyadenosine or deoxyguanosine, but thymidine is efficiently utilized. In this paper a new thymine-requiring mutant is described that differs from other thy- mutants (E. coli 15 T- and K12 T-) indicated previously in the relationship between the rate of DNA replication and the intracellular dTTP pool, and the mutant is able to phosphorylate deoxycytidine into deoxycytidine nucleotides during exponential growth. It also has considerable deoxycytidine kinase or such enzyme which can activate the phosphorylation of deoxycytidine. MATERIALS AND METHODS
Deoxyribonucleoside Metabolism in E. coli C thy- ura- Mutant
condly, uracil and thymine-requiring mutants were isolated after treatment with aminopterin 1171. Biological Assay
Bacteria are grown in synthetic medium containing casamino acid (0.5 %) supplemented with the requirements. The assay procedure for viable cell numbers, total protein synthesis, rates of ribonucleic acid synthesis, and deoxyribonucleic acid chain elongation by the incorporation of each ['4C]uracil and ['4C]thymine were based on a previous report [5]. Total DNA amounts and DNA synthesis in cultures with and without added dCyd (final concn 100nmol/ml) were measured with the diphenylamine reaction by Burton's method [18]. Deoxyadenosine 5'-monophosphate (dAMP) was used as a standard for the absolute amount of DNA. Preparations of Extracts
The preparation of the cell extracts was the same as described in a previous report [5]. The supernatants were used for the assays of thymidylate synthetase and deoxyribonucleoside kinase. The amount of protein was determined according to the method of Lowry et al. [19]. Bovine serum albumin (fraction V) was used as a control.
Chemicals
Uracil (Ura), thymine (Thy), thymidine (dThd), deoxycytidine (dCyd), deoxyuridine (dUrd), thymidine mono, di and triphosphates (dTMP, dTDP, dTTP), dTDP-glucose as the marker of dTDP-sugar, deoxycytidine mono, di and triphosphates (dCMP, dCDP, dCTP), deoxyuridine mono, di and triphosphates (dUMP, dUDP, dUTP) were purchased from Sigma Chemical Company Ltd. Casamino acid was from Difco Laboratories (Detroit, U.S.A.), and other chemicals were from Wako Pure Chemical Corporation Ltd (Japan). Isotopes
De~xy[S-~H]cytidine (specific activity20Ci/mmol), deo~y[5-~H]uridine monophosphate (specific activity 12.7 Ci/mmol), and [32P]orthophosphate(carrier free) were obtained from Nippon Isotope Corporation. Bacteria Escherichia coli C (wild type) and E. coli C thyuru- mutants isolated here from this E. coli C wild-type strain were named Thy A-11 and Thy A-35 mutants respectively. First, the uracil-requiring mutant was isolated after ultraviolet light irradiation and the penicillin screening method. Se-
Assay of Thymidylate Synthetase Activity
The activity of thymidylate synthetase was measured by the release of tritium from [5-3H]deoxyuridylate by the procedures of Lomax and Greenberg [20] and Roodman and Greenberg [21]. The relationship between concentration of protein and reaction rate is shown in Fig.1 for extracts of Thy A-11 and Thy A-35 cell cultures grown at 37°C for many generations and compared with the parent E. coli C strain. The average specific activity for E. coli C (wild type) is 4.35 units/mg, but we can measure almost no activity of thymidylate synthetase in these mutant cells. It is concluded that thy- uramutants (Thy A-11 and Thy A-35) are completely deficient in the activity of thymidylate synthetase. Measurement of Thymidine and Deoxycytidine Nucleotide Pools
The assay of intracellular thymidine nucleotide pools labeled with [3H]thymine in cultures with and without added dCyd (final concn 100 nmol/ml) were based on a previous report [5]. Deoxycytidine nucleotide pools were measured by the same procedure as the measurement of thymidine nucleotides with minor modification, using [3H]deoxycytidine instead of [3H]thymine.The cells were labeled with [3H]deoxy-
T. Ohkawa
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167
----g_.d.,*-.-c3--r--~--.C-rC-L-
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Protein (mg)
Fig. 1. Relutionship of' tritium releuse ussay,for thymidylate synthetase to amount of extract. Enzyme activity is expressed in units. 1 unit = 1 nmol of tritium atom released/min at 37'C. E. coli C (wild Thy A-I1 mutant ( x - - - - x ) and Thy A-3 type) (-a), mutant (A - - -A)
cytidine (specific activity 88.5 Ci/mol) by addition of an excess of unlabeled deoxyuridine (final concn 224.2 pg/ml). The chromatogram applied with the cell extracts and unlabeled carrier substances was developed once with distilled water to reject bases and nucleosides, and this chromatogram was also developed in the first dimension with a solution of 0.6 M LiCl in saturated boric acid. Prior to development in the second dimension with a solution of 1 M acetic acid in 3 M LiCl(9 :1) to separate deoxycytidine nucleotides from deoxyuridine nucleotides, boric acid and lithium chloride had to be removed according to the method of Randerath and Randerath [22]. After thin-layer chromatography, the level of acid soluble radioactive nucleotides was 85 - 88 % of the original. Quantitative Determination of Ac id-Soluble Nucleo t ides
The assay of intracellular acid-soluble nucleotides was carried out by the same procedure as the measurement of thymidine nucleotides, using [32P]orthophosphate (1.49 mM phosphate, 32P 100 pCi/ml) for two generations (2 h) instead of [3H]thymine [5].The chromatogram spotted with the cell extracts and unlabeled markers was developed as described by Neuhard [23] and Fuchs [24]. The radioactivity was measured as described previously [5]. Kinase Assays
The supernatants of cell extracts were adjusted to pH 4.2 with 1 .0 M HC1[25] and centrifuged at 0 "C. The precipitates dissolved in 0.1 M Tris-HCI buffer (pH 7.8) were used for the assay of deoxyribo-
nucleoside kinase. The assay was performed as described by Okazaki and Kornberg [26] and Karlstrom [15] with minor modifications. An incubation mixture of 0.24 ml contained 41 nmol 3H-labeled deoxyribonucleoside(15 pCi/mol), 30pmol p H 7.8 TrisHCl buffer, 20nmoles MgClz, 30nmol MnC12, 160 nmol ATP, 8 pg bovine serum albumin, and an extract (enzyme solution described above) containing 0.035 -0.085 mg protein. The mixture was incubated for 15, 30 or 60 min at 37 "C, and the reaction was terminated by adding 0.25 ml 10% ice-cold acetic acid (final concn 5%). These samples were dried by evaporation and were redissolved in 0.1 ml distilled water. A 10-p1 portion was applied to a thin-layer plate with unlabeled carrier substances, and the chromatogram was developed twice in the same dimension with distilled water to reject other reacted salts than deoxyribonucleotides. After the chromatography, the radioactivity was measured as described in a previous report [5]. The developing solvents were as follows : 1 M acetic acid/3 M LiCl(9il) [22] for deoxycytidine, and 0.45 M LiCl in saturated boric acid adjusted to pH 7.0 with 1 M N H 4 0 H [5] for thymidine. RESULTS D N A Replication and R N A Synthesis in E. coli C thy- ura- Mutants
In the thy- ura- mutants isolated here from E. coli C strain (wild type), when rates of DNA replication and RNA synthesis are measured by the incorporation of [14C]thymine and ['4C]uracil into the thy- ura- mutants during exponential growth, we find one mutant (Thy A-35) that performed the DNA replication at a rate only one-half that of the other mutant (Thy A-11). But the RNA synthesis in the Thy A-35 mutant shows the same rate as that of the Thy A-I 1 mutant (Fig. 2). These data show that, based on the relationship that the rate of RNA synthesis is associated with the rate of DNA replication in the Thy A-11 mutant during exponential growth, the rate of RNA synthesis is not associated with the rate of DNA replication in the Thy A-35 mutant. Viable Cell Numbers and Protein Synthesis in E. coli C thy- ura- Mutants
Most protein is made in the cytoplasm, and RNA, rather than DNA, is associated with its biosynthesis [27]. From the result shown in Fig.2, the kinetics of viable cell numbers and of the protein synthesis should be tested in the Thy A-35 mutant compared with those in the Thy A-11 mutant during exponential growth. Fig.3 shows that the increasing rate of viable cell numbers in the Thy A-35 mutant is at a slightly lower
Deoxyribonucleoside Metabolism in E. coli c tl7y- ura- Mutant
168
60
-
: ,50 F& ._
-
c
m L O
- 5E
40 -
3. 0
30 -
'3
2 5
Y
I
20
-
10 ,.
oov
3'0
60
do
;1
0
1&
Time (min)
30
60 90 Time (rnin)
120
150
Fig. 2. Kinetics of D N A replication and R N A synthesis by incorporations of ['4C]thymine and ['4C]uracil in both Thy A-11 and Thy A-35 mutants during exponential growth. Conditions and measurements of ratioactivities of acid-insoluble materials were as described in Materials and Methods. (A) DNA replications in both Thy A-I1 and Thy A-35 mutants; (B) RNA synthesis in both and Thy A-1 1 and Thy A-35 mutants. Thy A-I 1 mutant (+-O), Thy A-35 mutant ( x ---- x )
Fig. 3. Kinetics of viabilities and protein syntheses in both ThynA-ll and Thy A-35 niutants during exponential growth. Conditions were the same as those described in Fig.2. (A) Viable cell numbers in both Thy A-11 and Thy A-35 mutants, and E. coli C as a control. (B) Protein syntheses in both Thy A-11 and Thy A-35 mutants, and E. coli C as a control. Thy A-11 mutant (0-O), Thy A-35 mutant ( x x ), and E. coli C (wild type) (O----O)
level than that of the increasing rate in viable cell numbers of the Thy A-11 mutant, but the protein synthesis in the Thy A-35 mutant takes place'at the same rate as that of the protein synthesis in the Thy A-11 mutant. It is concluded that the rate of protein synthesis is consistent with that of RN A synthesis, but not the rate of DNA replication, in the Thy A-35 mutant.
replication in the Thy A-35 mutant (Fig.4B). Also, DNA replications in cultures of the Thy A-1 1 mutant with added dCyd and dUrd take place at the same rates as those in cultures without additions (Fig. 4A). Thymidine inhibits the DNA replications in both Thy A-11 and Thy A-35 mutants, as those mutant cells are sensitive to this compound. Also, to determine the exact DNA synthesis in the Thy A-35 mutant, the amount of DNA in the cell culture is chemically measured by the diphenylamine reaction of Burton's method (Fig.5 and Table 1). F i g 5 shows the DNA synthesis in E. colic (wild type) and thy- UYCI- mutants during exponential growth. The DNA synthesis in the Thy A-35 mutant shows a rate only one-half that in other strains (Fig. 5) and the DNA concentration (DNAjmg dry wt) in the Thy A-35 mutant is about a half of that in other strains (Table 1). Also, DNA synthesis and its concentration in the dCyd-added culture of the Thy A-35 mutant are the same as those in other strains. From these results, it is concluded that in the Thy A-35 mutant, in proportion to the reduced rate of DNA replication (or chain elongation), the DNA concentration and the differential rate of D NA synthesis decrease to a half those in other strains during exponential growth.
Effect of Deoxycytidine on D N A Replication or Synthesis in the Thy A-35 Mutant To investigate why DNA replication in the Thy A-35 mutant decreases to a rate only one-half that in the Thy A-I 1 mutant (Fig. 2) we first examined whether or not DNA replication in the Thy A-35 mutant recovers from its decreased rate by adding bases, ribonucleosides and deoxyribonucleosides. Fig. 4 shows that, by the incorporation of ['4C]thymine into the Thy A-35 mutant, the rate of DNA replication in the dCyd-added culture is increased two-fold compared to that in its culture without this addition, and is the same rate as that in the Thy A-I1 mutant. But deoxyuridine does not increase the rate of DNA
-
--
T. Ohkawa
169
1.01
A
,
0.02
I I
I
I
30
60
I
I
I
I
90 120 150 180 Time (min) Fig. 5. Kinetics of D N A syntheses in c~~iltures with and without added
0
deoxycytidine of’ both Thy A-I1 and Thy A-35 mutants during exponential growth. Conditions were the same as those described in Fig.2. Measurements of total DNA amounts in E. coli C (wild type) and these mutant strains were as described in Materials and Methods. dAMP was used as a standard for the absolute amount of DNA. E. coli C (wild type) (+----@), Thy A-I1 mutant (x----x), Thy A-11 mutant in culture with dCyd added (&----A), Thy A-35 mutant (*O), Thy A-35 mutant in dCyd-added culture (v----0)
Time (min)
Fig. 4. Kinetics of D N A replication by the incorporcrtion qf[14C]thymine in cultures of both Thy A-11 and Thj, A-35 mutants, with and without added deoxyribonucleosides, during exponential growth. Conditions were the same as those described in Fig. 2. (A) Thy A-11 mutant; (B) Thy A-35 mutant. Culture without added deoxydCyd added ( x x ), dUrd added ribonucleoside (---.); (A- - - A), dThd added (o----o) ~-
Thymidine Nucleotide Pools in Cultures of E. coli thy- ura- Mutants (Thy A-11 and Thy A-35) with and without Added Deoxycytidine It was reported previously that the decreased rate of DNA replication is based on a low dTTP pool during exponential growth in the pyrophosphorylase activated mutant (Ter-4) of E. coli K12 [ 5 ] . As it is surmised that the Thy A-35 mutant would have a smaller dTTP pool than that of the Thy A-1 1 mutant, we measured the intracellular thymidine nucleotide pools by the incorporation of [3H]thymine during exponential growth. Fig. 6 A and 7 A show intracellular pools of dTMP, dTDP, dTTP and dTDP-sugar in both Thy A-I1 and Thy A-35 mutants. dTMP, dTDP and dTTP pools in the Thy A-35 mutant increase about twofold compared to those pools for the Thy A-11 mutant. Also, the dTDP-sugar pool in the Thy A-35 mutant increases about 1.5-fold compared to that for the Thy A-1 1 mutant. These results provide
Table 1. Amounts of’ total D N A in E. coli C (wild type) and its thy- nrd- mutant strains Conditions were the same as those described in Fig.5. The cells were exponentially grown at 37 “C for 2 h and collected by centrifugation and washed with the synthetic medium. These samples were dried and measured dry weights of the cells and amounts of total DNA by Burton’s method [19]. dAMP was used as the standard for the absolute amount of DNA Bacterial strains
Deoxycytidine
E. coli C Thy A-11
-
Thy A-35
-
+ +
DNA DNA/mg dry wt 9.42 8.86 9.15 4.88 8.82
new evidence that intracellular thymidine nucleotide pools in the Thy A-35 mutant ( E . coli C thy- urastrain) differ from those in Ter mutants (Ter-4, Ter-15 and Ter-21) of the E. coli K12 strain [ 5 ] . In the Thy A-35 mutant, dTTP and dTDP pools in the dCyd-added culture increase about twofold compared to those in its culture without added dCyd and dTMP and dTDP-sugar pools in the dCydadded culture have the same value as those in its culture lacking this addition (Fig.7B). In the Thy A-11 mutant thymidine nucleotides pools in the dCyd-added culture have the same values as those in its culture without added dCyd (Fig.6B). It is con-
Deoxyribonucleoside Metabolism in E. coli C thy- ura- Mutant
170
A
-
h,
I-
/ -
30
60
90
0
30
Time (min)
60 Time (min)
90
Fig. 6. Kinetics of thymidine nucleotide syntheses in cultures with and without added cytidine of the 7 % A-11 ~ mutant during exponential growth. Conditions were the same as those described in Fig. 2. Thymidine nucleotides were measured as described in Materials and Methods. (A) Without added cytidine; (B) with added cytidine. dTTP (*-a), dTDP ( x - - - - x ) , dTMP (A---A), dTDP-sugar (0----0)
I
p,- -0--
,/'
0
30
60 Time (rnin)
90
"0
-
-
I
I
I
30
60
90
Time (min)
Fig. 7 . Kinetics of thymidine nucleotide .syntheses in cultures with and without udded deowycytirlinr (it rhc, 171>,A--j.i iiiut(uit during ewponential growth. Conditions and measurements of radioactivites were the same as those described in Fis. 6. (A) Without added deoxycytidine; (B) with added deoxycytidine. dTTP (0 --a), dTDP ( x---- x), dTMP (&---A), dTDP-sugar (0-- - -0)
cluded that DNA replication in the Thy A-35 mutant depends upon the intracellular dTTP pool when deoxycytidine is added to the culture. Also, with [32P]orthophosphate added to the culture, nucleoside triphosphate pools are measured in E. coli C (wild type) and its thy- U Y U - mutants during exponential growth. Table 2 shows eight nucleoside triphosphate pools after incubation for 2 h at 37 "C. Each nucleoside triphosphate in the Thy A-1 1
mutant has almost the same value as that of wild-type cells. In the Thy A-35 mutant purine rib0 and deoxyribonucleotides are of the same values as those for wild-type cells, but pyrimidine nucleotide increase 3 - 5-fold compared to those of the wild-type cells except for UTP. In particular, dTTP and dCTP pools in the dCyd-added culture increase about 1.5 - 2.0-fold compared to those in its culture without addition. Also, UTP in the Thy A-35 mutant is of almost the same
T. Ohkawa
171
Table 2. Nucleoside triphosphate pools of E. coli C (wild type) and its thy-ura- mutant strains Conditions were the same as those described in Fig. 6 [32P]Orthophosphate was incorporated into the cells during exponential growth for 2 h. Measurements of radioactive materials were as described in Materials and Methods p~~~~~~
Bacterial strains
Deoxycytidine
Nucleoside triphosphates -
-
-
-
ATP
-
-
~~
~
GTP
CTP
UTP
dATP
dGTP
~
dCTP
dTTP
~~
pmol x mi-' Ahbn unit-' ~ p - p
-
E. coli C Thy A-I1
~~~
189.6 230.8 211.5 224.1 261.6
-
+ +
Thy A-35
~~~~
-
75.3 95.4 86.1 81.4 115.3
22.4 23.9 24.8 60.5 70.7
Table 3. Incorporation of thymine and deoxycytidine into those deoxyribonucleotides in Thy A-11 and Thy A-35 mutants during exponential gro wth Conditions were the same as those described in Fig. 6. [3H]Thymine was incorporated into these mutants for 45 min, and [3H]deoxycytidine was incorporated into these mutants for 25 min in media with excess deoxyuridine added. Measurements of radioactive materials were as described in Materials and Methods Bacterial strains
Thymidine nucleotides
Deoxycytidine nucleotides ~
_
p
p
_
~
dTTP
dTDP
dTMP
dCTP
dCDP
dCMP
3.03 35.16
1.12 3.37
1.19 5.06
pmol x ml-' x Ahho unit-' Thy A-11 23.1 Thy A-35 118.2
5.87 22.67
36.9 209.6
~
~.
value as that in the wild-type cells, and does not show a marked increase in the dCyd-added culture. This UTP pool during exponential growth is now under investigation. Phosphorylution of Deoxycytidine in vivo and in vitro in the Thy A-35 Mutant
Table 3 shows deoxycytidine nucleotide pools produced after the addition of [3H]deoxycytidine in cultures of Thy A-11 and Thy A-35 mutants. dCTP in the Thy A-35 mutant has a 4-fold larger value than that in the Thy A-1 1 mutant, and dCDP and dCMP in the Thy A-35 mutant have about 1.5 - 2.0-fold larger values than those in the Thy A-11 mutant. Also, thymidine nucleotide pools in the Thy A-35 mutant increase about 5-fold in value compared to those in the Thy A-1 1 mutant when deoxycytidine is added to the culture. These data show that the increase of the dTTP pool in the Thy A-35 mutant accompanies the increase of the dCTP pool (Tables 2 and 3). According to works of Neuhard [23], Filpula and Fuchs [28] and Manwaring and Fuchs [29] in E. coli, dCTP shows a 1.0-2.0-fold larger value than dTTP using [32P]orthophosph.ate. In the Thy A-35 mutant, as the dTTP pool is very large, dCTP is a corre-
24.6 26.2 27.3 29.4 32.4
_
_
~
19.9 11.3 16.7 13.7 17.4
_ -
~ ~~~
4.89 4.62 4.72 8.24 9.43
~p
-
~
5.69 6.21 5.28 13.93 21.69
_
_
_
~
p
~
~
~~
~
~~
17.2 24.5 24.8 74.2 129.2
spondingly smaller pool than that of dTTP during incubation for 2 h using [32P]orthophosphate. But, as the dCTP pool in the Thy A-35 mutant after the addition of deoxycytidine increases 4-fold compared to that of the Thy A-11 mutant and E. coli C (wild type), it is considered that the Thy A-35 mutant is able to incorporate deoxycytidine as long as its concentration in the medium remains high enough. Measuring in vitro the phosphorylation of deoxycytidine by the pH 4.2 precipitate (which contained many kinds of enzymes) in crude extracts of the Thy A-11 and Thy A-35 mutants, with thymidine as the substrate, incorporation is proportional to the amount of extract added and to the incubation time. With deoxycytidine, 0.04 mg protein and 30 min incubation give the maximum incorporation values shown in Table 4. From the pH 4.2 precipitate of the extract in the Thy A-11 mutant, the produced dCMP is one-tenth the amount of dTMP. But in the Thy A-35 mutant, the amount of produced dCMP is about the same as that of dTMP. These data show that considerable deoxycytidine kinase or the enzyme which activates the phosphorylation of deoxycytidine is contained in the extracts of the Thy A-35 mutant.
DISCUSSION The intracellular dTTP pool exhibits two functions : one is a rate-limiting factor in the polymerization of DNA [5,6], and the other is a conversion to dTDPsugar by thymidine diphosphate glucose pyrophosphorylase for the biosynthesis of cell wall lipopolysaccharide [30]. Therefore in abnormal cell growth, the intracellular dTTP pool increases to a high value when the DNA elongation is inhibited by antibiotics (novobiocin and nalidixic acid) [24,31] or when the thimidine diphosphate glucose pyrophosphorylase is made defective by its gene mutation [5]. During the periods of thymine starvation in a thy- mutant ( E . coli K12T-U-Mp, 15T- and CR34), the dTTP
Deoxyribonucleoside Metabolism in E. coli C thy- ura- Mutant
172
Table 4. Phosphorylation of deoxyrihonucleosides by p H 4.2 precipitates in extructs of hoth Thy A-11 and Thy A-35 mutants Conditions were the same as those described in Fig.2. Enzyme assays were as described in Materials and Methods Bacterial strains
dNMP formed from
Deoxyribonucleoside added (dN) ~~-
dC
dT
nmol x min-' x mg protein-' ~~
Thy A-35
0.132
1.000
1.000
0.855
dC
~-
Deoxyribonucleoside
-
-
41.7
50.0
-
-
41.7
Deoxyribonucleoside monophosphate formed -~
~
dCMP
dU MP
nmol x min-' x mg protein-' Thy A-I1 Thy A-35
deoxycytidine deoxycytidine
1.000 1.000
dNMP
dNDP
dNTP
28.8 12.5 34.1 14.8
15.59 2.06 9.63 8.23
2.31 0.11 1.85 1.01
0.31 0.05 0.22 0.12
.
50.0
Table 5 Phoqhorylution and deaminution ofdeoxycytidine by p H 4 2 precipitatec in extracts of hoth T h j A-11 and Thy A-35 mutants Conditions were the same as those described in Table 4 Bacterial strdins
dN
nmol x mg protein-'
-~
Thy A-1 1
-
~~~~
~
dT
Sum of deoxyribonucleosides and deoxyribonucleotides formed after 30 min
1.046 0.561
pool decreases immediately to a very small value 1321, and in compensation the dCTP and dATP pools increase to very large values, but the dGTP pool was constant during incubation [23,31]. Also, the dTTP pool increases 1.5 - 6.0-fold compared to the original during uracil or cytidine starvation in the double pyrimidine-requiring mutant (uracil and cytidine auxotroph) [23]. These findings provide evidence that one pyrimidine nucleoside triphosphate (e.g. dCTP) pool accumulates during the inhibition of DNA synthesis while the other pyrimidine nucleoside triphosphate (e.g. dTTP) decreases, and vice versa. From these results, in the Thy A-35 mutant, the dTTP pool may increase to a large value as the dCTP pool decreases to a small value during exponential growth. This reduction of the dCTP pool causes a decreased rate of DNA replication. As a result, this reduced DNA replication reduces the DNA concentration (Table 1) as reported by Pritchard [4] and Pritchard and Zaritsky [3]. Although the rate of DNA synthesis in the dCyd-added culture increases twofold compared to that in its culture without addition (Fig.5), the dTTP pool in the dCyd-added culture does not decrease to one-half but increases to twice that of its culture without added dCyd. These data show that the DNA synthesis is absolutely dependent upon the dTTP pool.
As no rhamnose residue is detected in the core oligosaccharide of lipopolysaccharide of the E. coli C strain 1331, the dTDP-rhamnose 1341, which is a component of dTDP-sugar, would not be utilized for the lipopolysaccharide biosynthesis. From this, dTDPsugar pools in Thy A-1 1 and Thy A-35 mutants would show very high values during exponential growth. Comparing the data for the nucleotide pool in E. coli C and its mutant (Thy A-11 and Thy A-35) strains with those in other strains [23,24,28,29], in E. coli C and its mutant cells, pyrimidine ribonucleoside triphosphate (CTP and UTP) pools show much lower values than those of purine ribonucleoside triphosphate (ATP and GTP) pools, though the pyrimidine ribonucleoside triphosphate pools are of the same values as those of the purine ribonucleoside triphosphate pools in other strains (E. coli B and 15T-). As Thy A-11 and Thy A-35 mutants require uracil for growth, after the addition of uracil to the medium, the UTP pool in those mutants increases somewhat compared to the value of the UTP pool in E. coli C. In particular, the decrease of the UTP pool in the Thy A-35 mutant may give rise to the increase of other pyrimidine nucleotide (CTP, dCTP and dTTP) pools, but the addition of deoxycytidine without uridine and deoxyuridine increases the dTTP and dCTP pools, along with the rate of DNA replication during exponential growth. As the UTP pool does not increase by the addition of deoxycytidine, these results seem to point to mechanisms for the synthesis of pyrimidine nucleotide other than the decrease of the uridine nucleotide pool. Both lactobacilli [lo] and mammalian systems [l I ] can utilize deoxyribonucleosides for DNA synthesis, but E. coli cannot efficiently incorporate deoxyribonucleosides into DNA as these compounds are rapidly catabolized in the cells. Also, the mutant (OK 441) in the E. coli B which lacks four deoxyribonucleoside-catabolizing enzymes [ 161 did not incorporate any deoxyribonucleoside other than thymidine into its DNA, and only thymidine kinase
T. Ohkawa
but no other deoxyribonucleoside kinase could be detected in extracts of the mutant [15]. In Salmonella typhymurium, its cytidine (or deoxycytidine) deaminaseless mutant did not contain deoxycytidine kinase [23]. In the Thy A-35 mutant, DNA replication in the dCyd-added culture increases twofold compared to its rate in the culture without added dCyd, but the addition of other deoxyribonucleosides (deoxyadenosine, deoxyguanosine and deoxyuridine) than deoxycytidine and thymidine do not increase the rate of DNA replication. This result does not mean that the added deoxycytidine is first deaminated into deoxyuridine, after which DNA replication increases twofold. It is considered that the added deoxycytidine would be phosphorylated in the Thy A-35 mutant. As a result, Tables 3 and 4 show that the phosphorylation of deoxycytidine takes place in vivo and in vitro. These data indicate that extracts of the Thy A-35 mutant contain both thymidine kinase and considerable deoxycytidine kinase. or the enzyme which is able to phosphorylate deoxycytidine. It is concluded that the Thy A-35 mutant cells isolated from the E. coli C strain differ from the mutant cells from the E. coli B strain that were reported by Karlstrom [15]. As the activity of the deoxycytidine-catabolizing enzyme (deoxycytidine deaminase) is induced by the addition of deoxycytidine during exponential growth in E. coli [35], we must discuss the deamination of deoxycytidine added to the Media of Thy A-11 and Thy A-35 mutants. Deoxyuridine nucleotide pools show in vivo larger values ( 2 - 10-fold) than those of deoxycytidine nucleotide pools labeled with [3H]deoxycytidine during incubation for 45 min in these mutants. Therefore we performed the experiment to measure deoxycytidine nucleotide pools labeled with [3H]deoxycytidine in media containing excess deoxyuridine added in these mutants. Deoxyuridine nucleotide pools labeled with 3H show values one-half those of [3H]deoxycytidine nucleotide pools during incubation for 25 min (Table 3). Also, by the pH 4.2 precipitates purified partially in extracts of the Thy A-11 mutant, dUMP and dCMP are produced in the same amounts; but by the pH 4.2 precipitates in extracts of the Thy A-35 mutant, the amount of dUMP is only one-half that of dCMP (Table 5). These data present the possibility that considerable deoxycytidine kinase or the enzyme which is able to activate the phosphorylation of deoxycytidine could be purified from the extracts of the Thy A-35 mutant. As the deoxycytidine kinase has not been detected until now in E. coli B [15] and S. tjyhimurium [35], deoxycytidine may be phosphorylated by the enzyme which phosphorylates deoxyribonucleosides other than de-
173
oxycytidine in extracts of the Thy A-35 mutant. The purification of the enzyme and the phosphorylation of deoxycytidine in the Thy A-35 mutant are now under investigation. REFERENCES 1. Pritchard, R . H. & Zaritsky, A. (1970 Nature (Lond.) 226, 126 - 131. 2. Zaritsky, A. &Pritchard, R. H. (1971) J . Mol. Biol. 60, 65-74. 3. Zaritsky, A. & Pritchard, R. H. (1973) J . Bacteriol. 114, 824837. 4. Pritchard, R. H. (1974) Philos. Trans. R. Soc. L,ond. B, Biol. Sci. 267, 303 - 336. 5. Ohkawa, T. (1976) Eur. J . Biochem. 61, 81-91. 6. Beacham, I. R., Beacham, K., Zaritsky, A. Sr Pritchard, R. H. (1971) J . Mol. B i d . 60, 75-86. 7. Rachmeler, M., Gerhart, J. & Rosmerm, J . (1961). Biophys. Acta 49, 222 - 225. 8. Fangman, W. L. (1969) J . Bacteriol. 99, 681 -687. 9. Fangman, W. L. & Novick, A. (1966) J . Bacteriol. 91, 23902394. 10. Hoff-Jorgensen, E. (1952) Biochrm. J . 50,400-403. 11. Reichard, P. (1957) Acta Chem. Scand. 11, 11 -16. 12. Cohen, S. S. & Barner, H. D . (1957) J . B i d . Chem. 226, 631 64L. 13. Koch, A. L. & Vallee, G . (1959) J . Bid. Chem. 234, 12131228. 14. Manson, L. A. & Lampen, J. 0. (1951) J . B i d . Chem. 193, 539 - 549. 25. Karlstrom, H. 0. (1970) Eur. J . Biochem. 17, 68-71. 16. Karlstrom, H. 0. (1968) J . Buctrriol. 95, 1069-1077. 17. Stacev. K. A. & Simon. E. (1965)J. Bacteriol. 90. 554-555. 18. Burton, K. (1956) Biochem: J . 62, 315-323. 19. Lowry, 0. H., Rosebrough, N. J., F a n , A. L. & Randal, R. J. (1951) J . Mol. Biol. 193, 265-275. 20. Lomax, M. I. S. L Greenberg, G. R. (1967) J . B i d . Chem. 242, 109-113. 21. Roodman, S . T. & Greenberg, G. R. (1971) J . B i d . Chem. 246, 2699-2617. 22. Randerath, K. & Randerath, E. (1965) Anal. Biochem. 13, 575 - 579. 23. Neuhard, J. (1968) J . Bacteriol. 96, 1519-1527. 24. Fuchs, J . A. (1977) J . Bacteriol. 130, 957-959. 25. Bessman, M. J. (1963) Methods Enzymol. 6 , 166-176. 26. Okazaki, R. & Kornberg, A. (1964) J . B i d . Chem. 239, 269274. 27. Brachet, J. (1960) The Biological Role of Ribonucleic Acid, Elsevier, Amsterdam. 28. Filpula, D. & Fuchs, J. A. (1977) J . Bacteriol. 130, 107-113. 29. Manwaring, J . D. & Fuchs, J. A. (1977) J . Bacteriol. 130, 960 - 962. 30. Liideritz, O., Staub, A. M. & Westphal, 0. (1966) Bacteriol. Rev.30, 192-255. 31. Neuhard, J. & Thomassen, E. (1971) Eur, J . Biochem. 20, 36 - 43. 32. Ohkawa, T. (1975) Eur. J . Biochem. 60, 57-66. 33. Feige, U. & Stirm, S. (1976) Biochem. Biophys. Res. Commun. 71,566-573. 34. Okazaki, R., Okazaki, T., Strominger, Y. L. & Michelson, A. M. (1962) J . B i d . Chem. 237, 3014-3026. 35. O’Donovan, G. A. L Neuhard, J. (1970) Bacteriol. Rev. 34, 278 - 343.
T. Ohkawa, Department of Biochemistry, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa-shi, Ishikawa-ken, Japan 920
Abnormal Metabolism of Thymidine Nucleotides and Phosphorylation of Deoxycytidine in Escherichia coli C thy- ura- Mutant Tsutomu OHKAWA Department of Biochemistry, Kanazawa University School of Medicine (Received July 10, 1978 / J u n e 12, 1979)
The Escherichia coli C thy- ura- mutant (Thy A-35) shows a rate of DNA replication (or chain elongation) one-half that of the other thy- ura- mutant (Thy A-1 l), but RNA and protein syntheses in the Thy A-35 mutant show the same rates as those in the Thy A-1 1 mutant. The viable cell numbers in the Thy A-35 mutant exhibit a slightly slower rate than those in the Thy A-1 1 mutant. Thymidine and deoxycytidine triphosphate (dTTP and dCTP) pools in the Thy A-35 mutant increase 2 - 3-fold compared to those in the Thy A-1 1 mutant during exponential growth. As shown by Pritchard and Lond. B. Biol. Sci. (1974) 267, Zaritsky [J. Bacteriol. (1973) 114, 824-837; Philos. Trans. R. SOC. 303 - 3361 the reduced rate of DNA replication in the Thy A-35 mutant reduces the DNA concentration. Consequently, the differential rate of DNA synthesis in the Thy A-35 mutant is lower than that in other strains. Also, in the Thy A-35 mutant the rate of DNA replication in the culture with deoxycytidine added increases twofold compared to that in the culture without any addition, and this rate is the same as that of DNA replication in the Thy A-11 mutant. The thymidine triphosphate (dTTP) pool in the culture with deoxycytidine of the Thy A-35 mutant increases twofold compared to its value in the culture lacking deoxycytidine. It is concluded that DNA replications in both Thy A-1 1 and Thy A-35 mutants depend upon the intracellular dTTP pool. In vivo and in vitro the Thy A-35. mutant cells are able to phosphorylate deoxycytidine into deoxycytidine nucleotides, and have considerable deoxycytidine kinase or such enzyme which can activate the phosphorylation of deoxycytidine. For the purpose of studying the relationship between the thymidine triphosphate (dTTP) pool and deoxyribonucleic acid metabolism in E. coli, the thymine-requiring mutant is generally isolated. Recently it was reported that the replication time of the chromosome can be varied without a concomitant change in growth rate, simply by changing the concentration of thymine in the growth medium in thy- strains of E. coli [l - 31. In particular, when the thymine concentration in the growth medium of a thy- strain is reduced, the rate of DNA elongation is also reduced [I]. This effect reduces the average DNA concentration (the DNA : mass ratio). An increase Enzymes. Thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase (EC 2.4.2.4) ; thymidine kinase or ATP: thymine 5’-phosphotransferase (EC 2.7.1.21); cytidine (deoxycytidine) deaminase or cytidine (deoxycytidine) aminohydrolase (EC 3.5.4.5); cytidine (deoxycytidine) kinase or ATP:cytidine (deoxycytidine) 5’-phosphotransferase (EC 2.7.1.74); thymidylate synthase (EC 2.1.1.45).
in the growth rate of a thy+ culture has exactly the same effect on DNA concentration [3,4]. One mutant (Thy A-35) isolated here from E . coli C strain by aminopterin treatment shows a rate of DNA replication lower than that of other mutants under the same conditions. Therefore, it is considered that the average DNA concentration in the Thy A-35 mutant would be reduced more than that in other mutants. From this, it is surmised that the dTTP pool in the Thy A-35 mutant would be reduced more than that in other mutants, because the rate of DNA replication depends upon the size of the dTTP pool in thymutants of E. coli 15 T- and K12 T - [5,6]. Exogenous thymidine is readily incorporated into DNA in wild-type E. coli cells, but the incorporation soon ceases after a short time, as the enzyme thymidine phosphorylase degrades thymidine to thymine and deoxyribose I-phosphate [7]. Accordingly, mutants lacking this enzyme were able to incorporate thymidine for extended periods of time [8,9]. It is often assumed
166
that E. coli can incorporate deoxyribonucleosides other than thymidine as long as their concentrations in the medium are high enough, because both lactobacilli [lo] and mammalian systems [11] can utilize other deoxyribonucleosides for DNA synthesis. But the other deoxyribonucleosides have not been directly incorporated into DNA, as these compounds are rapidly catabolized in wild-type E. coli [12- 141. Karlstrom [15] has recently isolated the mutant (OK 441) that lacks four deoxyribonucleoside-catabolizing enzymes [16] to measure directly the incorporation of four deoxyribonucleosides. This mutant (OK 441) is unable to incorporate significant amounts of deoxycytidine, deoxyadenosine or deoxyguanosine, but thymidine is efficiently utilized. In this paper a new thymine-requiring mutant is described that differs from other thy- mutants (E. coli 15 T- and K12 T-) indicated previously in the relationship between the rate of DNA replication and the intracellular dTTP pool, and the mutant is able to phosphorylate deoxycytidine into deoxycytidine nucleotides during exponential growth. It also has considerable deoxycytidine kinase or such enzyme which can activate the phosphorylation of deoxycytidine. MATERIALS AND METHODS
Deoxyribonucleoside Metabolism in E. coli C thy- ura- Mutant
condly, uracil and thymine-requiring mutants were isolated after treatment with aminopterin 1171. Biological Assay
Bacteria are grown in synthetic medium containing casamino acid (0.5 %) supplemented with the requirements. The assay procedure for viable cell numbers, total protein synthesis, rates of ribonucleic acid synthesis, and deoxyribonucleic acid chain elongation by the incorporation of each ['4C]uracil and ['4C]thymine were based on a previous report [5]. Total DNA amounts and DNA synthesis in cultures with and without added dCyd (final concn 100nmol/ml) were measured with the diphenylamine reaction by Burton's method [18]. Deoxyadenosine 5'-monophosphate (dAMP) was used as a standard for the absolute amount of DNA. Preparations of Extracts
The preparation of the cell extracts was the same as described in a previous report [5]. The supernatants were used for the assays of thymidylate synthetase and deoxyribonucleoside kinase. The amount of protein was determined according to the method of Lowry et al. [19]. Bovine serum albumin (fraction V) was used as a control.
Chemicals
Uracil (Ura), thymine (Thy), thymidine (dThd), deoxycytidine (dCyd), deoxyuridine (dUrd), thymidine mono, di and triphosphates (dTMP, dTDP, dTTP), dTDP-glucose as the marker of dTDP-sugar, deoxycytidine mono, di and triphosphates (dCMP, dCDP, dCTP), deoxyuridine mono, di and triphosphates (dUMP, dUDP, dUTP) were purchased from Sigma Chemical Company Ltd. Casamino acid was from Difco Laboratories (Detroit, U.S.A.), and other chemicals were from Wako Pure Chemical Corporation Ltd (Japan). Isotopes
De~xy[S-~H]cytidine (specific activity20Ci/mmol), deo~y[5-~H]uridine monophosphate (specific activity 12.7 Ci/mmol), and [32P]orthophosphate(carrier free) were obtained from Nippon Isotope Corporation. Bacteria Escherichia coli C (wild type) and E. coli C thyuru- mutants isolated here from this E. coli C wild-type strain were named Thy A-11 and Thy A-35 mutants respectively. First, the uracil-requiring mutant was isolated after ultraviolet light irradiation and the penicillin screening method. Se-
Assay of Thymidylate Synthetase Activity
The activity of thymidylate synthetase was measured by the release of tritium from [5-3H]deoxyuridylate by the procedures of Lomax and Greenberg [20] and Roodman and Greenberg [21]. The relationship between concentration of protein and reaction rate is shown in Fig.1 for extracts of Thy A-11 and Thy A-35 cell cultures grown at 37°C for many generations and compared with the parent E. coli C strain. The average specific activity for E. coli C (wild type) is 4.35 units/mg, but we can measure almost no activity of thymidylate synthetase in these mutant cells. It is concluded that thy- uramutants (Thy A-11 and Thy A-35) are completely deficient in the activity of thymidylate synthetase. Measurement of Thymidine and Deoxycytidine Nucleotide Pools
The assay of intracellular thymidine nucleotide pools labeled with [3H]thymine in cultures with and without added dCyd (final concn 100 nmol/ml) were based on a previous report [5]. Deoxycytidine nucleotide pools were measured by the same procedure as the measurement of thymidine nucleotides with minor modification, using [3H]deoxycytidine instead of [3H]thymine.The cells were labeled with [3H]deoxy-
T. Ohkawa
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0
0.1
0.2
0.3
0.4
0.5
0.6
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Protein (mg)
Fig. 1. Relutionship of' tritium releuse ussay,for thymidylate synthetase to amount of extract. Enzyme activity is expressed in units. 1 unit = 1 nmol of tritium atom released/min at 37'C. E. coli C (wild Thy A-I1 mutant ( x - - - - x ) and Thy A-3 type) (-a), mutant (A - - -A)
cytidine (specific activity 88.5 Ci/mol) by addition of an excess of unlabeled deoxyuridine (final concn 224.2 pg/ml). The chromatogram applied with the cell extracts and unlabeled carrier substances was developed once with distilled water to reject bases and nucleosides, and this chromatogram was also developed in the first dimension with a solution of 0.6 M LiCl in saturated boric acid. Prior to development in the second dimension with a solution of 1 M acetic acid in 3 M LiCl(9 :1) to separate deoxycytidine nucleotides from deoxyuridine nucleotides, boric acid and lithium chloride had to be removed according to the method of Randerath and Randerath [22]. After thin-layer chromatography, the level of acid soluble radioactive nucleotides was 85 - 88 % of the original. Quantitative Determination of Ac id-Soluble Nucleo t ides
The assay of intracellular acid-soluble nucleotides was carried out by the same procedure as the measurement of thymidine nucleotides, using [32P]orthophosphate (1.49 mM phosphate, 32P 100 pCi/ml) for two generations (2 h) instead of [3H]thymine [5].The chromatogram spotted with the cell extracts and unlabeled markers was developed as described by Neuhard [23] and Fuchs [24]. The radioactivity was measured as described previously [5]. Kinase Assays
The supernatants of cell extracts were adjusted to pH 4.2 with 1 .0 M HC1[25] and centrifuged at 0 "C. The precipitates dissolved in 0.1 M Tris-HCI buffer (pH 7.8) were used for the assay of deoxyribo-
nucleoside kinase. The assay was performed as described by Okazaki and Kornberg [26] and Karlstrom [15] with minor modifications. An incubation mixture of 0.24 ml contained 41 nmol 3H-labeled deoxyribonucleoside(15 pCi/mol), 30pmol p H 7.8 TrisHCl buffer, 20nmoles MgClz, 30nmol MnC12, 160 nmol ATP, 8 pg bovine serum albumin, and an extract (enzyme solution described above) containing 0.035 -0.085 mg protein. The mixture was incubated for 15, 30 or 60 min at 37 "C, and the reaction was terminated by adding 0.25 ml 10% ice-cold acetic acid (final concn 5%). These samples were dried by evaporation and were redissolved in 0.1 ml distilled water. A 10-p1 portion was applied to a thin-layer plate with unlabeled carrier substances, and the chromatogram was developed twice in the same dimension with distilled water to reject other reacted salts than deoxyribonucleotides. After the chromatography, the radioactivity was measured as described in a previous report [5]. The developing solvents were as follows : 1 M acetic acid/3 M LiCl(9il) [22] for deoxycytidine, and 0.45 M LiCl in saturated boric acid adjusted to pH 7.0 with 1 M N H 4 0 H [5] for thymidine. RESULTS D N A Replication and R N A Synthesis in E. coli C thy- ura- Mutants
In the thy- ura- mutants isolated here from E. coli C strain (wild type), when rates of DNA replication and RNA synthesis are measured by the incorporation of [14C]thymine and ['4C]uracil into the thy- ura- mutants during exponential growth, we find one mutant (Thy A-35) that performed the DNA replication at a rate only one-half that of the other mutant (Thy A-11). But the RNA synthesis in the Thy A-35 mutant shows the same rate as that of the Thy A-I 1 mutant (Fig. 2). These data show that, based on the relationship that the rate of RNA synthesis is associated with the rate of DNA replication in the Thy A-11 mutant during exponential growth, the rate of RNA synthesis is not associated with the rate of DNA replication in the Thy A-35 mutant. Viable Cell Numbers and Protein Synthesis in E. coli C thy- ura- Mutants
Most protein is made in the cytoplasm, and RNA, rather than DNA, is associated with its biosynthesis [27]. From the result shown in Fig.2, the kinetics of viable cell numbers and of the protein synthesis should be tested in the Thy A-35 mutant compared with those in the Thy A-11 mutant during exponential growth. Fig.3 shows that the increasing rate of viable cell numbers in the Thy A-35 mutant is at a slightly lower
Deoxyribonucleoside Metabolism in E. coli c tl7y- ura- Mutant
168
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: ,50 F& ._
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oov
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do
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Fig. 2. Kinetics of D N A replication and R N A synthesis by incorporations of ['4C]thymine and ['4C]uracil in both Thy A-11 and Thy A-35 mutants during exponential growth. Conditions and measurements of ratioactivities of acid-insoluble materials were as described in Materials and Methods. (A) DNA replications in both Thy A-I1 and Thy A-35 mutants; (B) RNA synthesis in both and Thy A-1 1 and Thy A-35 mutants. Thy A-I 1 mutant (+-O), Thy A-35 mutant ( x ---- x )
Fig. 3. Kinetics of viabilities and protein syntheses in both ThynA-ll and Thy A-35 niutants during exponential growth. Conditions were the same as those described in Fig.2. (A) Viable cell numbers in both Thy A-11 and Thy A-35 mutants, and E. coli C as a control. (B) Protein syntheses in both Thy A-11 and Thy A-35 mutants, and E. coli C as a control. Thy A-11 mutant (0-O), Thy A-35 mutant ( x x ), and E. coli C (wild type) (O----O)
level than that of the increasing rate in viable cell numbers of the Thy A-11 mutant, but the protein synthesis in the Thy A-35 mutant takes place'at the same rate as that of the protein synthesis in the Thy A-11 mutant. It is concluded that the rate of protein synthesis is consistent with that of RN A synthesis, but not the rate of DNA replication, in the Thy A-35 mutant.
replication in the Thy A-35 mutant (Fig.4B). Also, DNA replications in cultures of the Thy A-1 1 mutant with added dCyd and dUrd take place at the same rates as those in cultures without additions (Fig. 4A). Thymidine inhibits the DNA replications in both Thy A-11 and Thy A-35 mutants, as those mutant cells are sensitive to this compound. Also, to determine the exact DNA synthesis in the Thy A-35 mutant, the amount of DNA in the cell culture is chemically measured by the diphenylamine reaction of Burton's method (Fig.5 and Table 1). F i g 5 shows the DNA synthesis in E. colic (wild type) and thy- UYCI- mutants during exponential growth. The DNA synthesis in the Thy A-35 mutant shows a rate only one-half that in other strains (Fig. 5) and the DNA concentration (DNAjmg dry wt) in the Thy A-35 mutant is about a half of that in other strains (Table 1). Also, DNA synthesis and its concentration in the dCyd-added culture of the Thy A-35 mutant are the same as those in other strains. From these results, it is concluded that in the Thy A-35 mutant, in proportion to the reduced rate of DNA replication (or chain elongation), the DNA concentration and the differential rate of D NA synthesis decrease to a half those in other strains during exponential growth.
Effect of Deoxycytidine on D N A Replication or Synthesis in the Thy A-35 Mutant To investigate why DNA replication in the Thy A-35 mutant decreases to a rate only one-half that in the Thy A-I 1 mutant (Fig. 2) we first examined whether or not DNA replication in the Thy A-35 mutant recovers from its decreased rate by adding bases, ribonucleosides and deoxyribonucleosides. Fig. 4 shows that, by the incorporation of ['4C]thymine into the Thy A-35 mutant, the rate of DNA replication in the dCyd-added culture is increased two-fold compared to that in its culture without this addition, and is the same rate as that in the Thy A-I1 mutant. But deoxyuridine does not increase the rate of DNA
-
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T. Ohkawa
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90 120 150 180 Time (min) Fig. 5. Kinetics of D N A syntheses in c~~iltures with and without added
0
deoxycytidine of’ both Thy A-I1 and Thy A-35 mutants during exponential growth. Conditions were the same as those described in Fig.2. Measurements of total DNA amounts in E. coli C (wild type) and these mutant strains were as described in Materials and Methods. dAMP was used as a standard for the absolute amount of DNA. E. coli C (wild type) (+----@), Thy A-I1 mutant (x----x), Thy A-11 mutant in culture with dCyd added (&----A), Thy A-35 mutant (*O), Thy A-35 mutant in dCyd-added culture (v----0)
Time (min)
Fig. 4. Kinetics of D N A replication by the incorporcrtion qf[14C]thymine in cultures of both Thy A-11 and Thj, A-35 mutants, with and without added deoxyribonucleosides, during exponential growth. Conditions were the same as those described in Fig. 2. (A) Thy A-11 mutant; (B) Thy A-35 mutant. Culture without added deoxydCyd added ( x x ), dUrd added ribonucleoside (---.); (A- - - A), dThd added (o----o) ~-
Thymidine Nucleotide Pools in Cultures of E. coli thy- ura- Mutants (Thy A-11 and Thy A-35) with and without Added Deoxycytidine It was reported previously that the decreased rate of DNA replication is based on a low dTTP pool during exponential growth in the pyrophosphorylase activated mutant (Ter-4) of E. coli K12 [ 5 ] . As it is surmised that the Thy A-35 mutant would have a smaller dTTP pool than that of the Thy A-1 1 mutant, we measured the intracellular thymidine nucleotide pools by the incorporation of [3H]thymine during exponential growth. Fig. 6 A and 7 A show intracellular pools of dTMP, dTDP, dTTP and dTDP-sugar in both Thy A-I1 and Thy A-35 mutants. dTMP, dTDP and dTTP pools in the Thy A-35 mutant increase about twofold compared to those pools for the Thy A-11 mutant. Also, the dTDP-sugar pool in the Thy A-35 mutant increases about 1.5-fold compared to that for the Thy A-1 1 mutant. These results provide
Table 1. Amounts of’ total D N A in E. coli C (wild type) and its thy- nrd- mutant strains Conditions were the same as those described in Fig.5. The cells were exponentially grown at 37 “C for 2 h and collected by centrifugation and washed with the synthetic medium. These samples were dried and measured dry weights of the cells and amounts of total DNA by Burton’s method [19]. dAMP was used as the standard for the absolute amount of DNA Bacterial strains
Deoxycytidine
E. coli C Thy A-11
-
Thy A-35
-
+ +
DNA DNA/mg dry wt 9.42 8.86 9.15 4.88 8.82
new evidence that intracellular thymidine nucleotide pools in the Thy A-35 mutant ( E . coli C thy- urastrain) differ from those in Ter mutants (Ter-4, Ter-15 and Ter-21) of the E. coli K12 strain [ 5 ] . In the Thy A-35 mutant, dTTP and dTDP pools in the dCyd-added culture increase about twofold compared to those in its culture without added dCyd and dTMP and dTDP-sugar pools in the dCydadded culture have the same value as those in its culture lacking this addition (Fig.7B). In the Thy A-11 mutant thymidine nucleotides pools in the dCyd-added culture have the same values as those in its culture without added dCyd (Fig.6B). It is con-
Deoxyribonucleoside Metabolism in E. coli C thy- ura- Mutant
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Fig. 6. Kinetics of thymidine nucleotide syntheses in cultures with and without added cytidine of the 7 % A-11 ~ mutant during exponential growth. Conditions were the same as those described in Fig. 2. Thymidine nucleotides were measured as described in Materials and Methods. (A) Without added cytidine; (B) with added cytidine. dTTP (*-a), dTDP ( x - - - - x ) , dTMP (A---A), dTDP-sugar (0----0)
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I
I
I
30
60
90
Time (min)
Fig. 7 . Kinetics of thymidine nucleotide .syntheses in cultures with and without udded deowycytirlinr (it rhc, 171>,A--j.i iiiut(uit during ewponential growth. Conditions and measurements of radioactivites were the same as those described in Fis. 6. (A) Without added deoxycytidine; (B) with added deoxycytidine. dTTP (0 --a), dTDP ( x---- x), dTMP (&---A), dTDP-sugar (0-- - -0)
cluded that DNA replication in the Thy A-35 mutant depends upon the intracellular dTTP pool when deoxycytidine is added to the culture. Also, with [32P]orthophosphate added to the culture, nucleoside triphosphate pools are measured in E. coli C (wild type) and its thy- U Y U - mutants during exponential growth. Table 2 shows eight nucleoside triphosphate pools after incubation for 2 h at 37 "C. Each nucleoside triphosphate in the Thy A-1 1
mutant has almost the same value as that of wild-type cells. In the Thy A-35 mutant purine rib0 and deoxyribonucleotides are of the same values as those for wild-type cells, but pyrimidine nucleotide increase 3 - 5-fold compared to those of the wild-type cells except for UTP. In particular, dTTP and dCTP pools in the dCyd-added culture increase about 1.5 - 2.0-fold compared to those in its culture without addition. Also, UTP in the Thy A-35 mutant is of almost the same
T. Ohkawa
171
Table 2. Nucleoside triphosphate pools of E. coli C (wild type) and its thy-ura- mutant strains Conditions were the same as those described in Fig. 6 [32P]Orthophosphate was incorporated into the cells during exponential growth for 2 h. Measurements of radioactive materials were as described in Materials and Methods p~~~~~~
Bacterial strains
Deoxycytidine
Nucleoside triphosphates -
-
-
-
ATP
-
-
~~
~
GTP
CTP
UTP
dATP
dGTP
~
dCTP
dTTP
~~
pmol x mi-' Ahbn unit-' ~ p - p
-
E. coli C Thy A-I1
~~~
189.6 230.8 211.5 224.1 261.6
-
+ +
Thy A-35
~~~~
-
75.3 95.4 86.1 81.4 115.3
22.4 23.9 24.8 60.5 70.7
Table 3. Incorporation of thymine and deoxycytidine into those deoxyribonucleotides in Thy A-11 and Thy A-35 mutants during exponential gro wth Conditions were the same as those described in Fig. 6. [3H]Thymine was incorporated into these mutants for 45 min, and [3H]deoxycytidine was incorporated into these mutants for 25 min in media with excess deoxyuridine added. Measurements of radioactive materials were as described in Materials and Methods Bacterial strains
Thymidine nucleotides
Deoxycytidine nucleotides ~
_
p
p
_
~
dTTP
dTDP
dTMP
dCTP
dCDP
dCMP
3.03 35.16
1.12 3.37
1.19 5.06
pmol x ml-' x Ahho unit-' Thy A-11 23.1 Thy A-35 118.2
5.87 22.67
36.9 209.6
~
~.
value as that in the wild-type cells, and does not show a marked increase in the dCyd-added culture. This UTP pool during exponential growth is now under investigation. Phosphorylution of Deoxycytidine in vivo and in vitro in the Thy A-35 Mutant
Table 3 shows deoxycytidine nucleotide pools produced after the addition of [3H]deoxycytidine in cultures of Thy A-11 and Thy A-35 mutants. dCTP in the Thy A-35 mutant has a 4-fold larger value than that in the Thy A-1 1 mutant, and dCDP and dCMP in the Thy A-35 mutant have about 1.5 - 2.0-fold larger values than those in the Thy A-11 mutant. Also, thymidine nucleotide pools in the Thy A-35 mutant increase about 5-fold in value compared to those in the Thy A-1 1 mutant when deoxycytidine is added to the culture. These data show that the increase of the dTTP pool in the Thy A-35 mutant accompanies the increase of the dCTP pool (Tables 2 and 3). According to works of Neuhard [23], Filpula and Fuchs [28] and Manwaring and Fuchs [29] in E. coli, dCTP shows a 1.0-2.0-fold larger value than dTTP using [32P]orthophosph.ate. In the Thy A-35 mutant, as the dTTP pool is very large, dCTP is a corre-
24.6 26.2 27.3 29.4 32.4
_
_
~
19.9 11.3 16.7 13.7 17.4
_ -
~ ~~~
4.89 4.62 4.72 8.24 9.43
~p
-
~
5.69 6.21 5.28 13.93 21.69
_
_
_
~
p
~
~
~~
~
~~
17.2 24.5 24.8 74.2 129.2
spondingly smaller pool than that of dTTP during incubation for 2 h using [32P]orthophosphate. But, as the dCTP pool in the Thy A-35 mutant after the addition of deoxycytidine increases 4-fold compared to that of the Thy A-11 mutant and E. coli C (wild type), it is considered that the Thy A-35 mutant is able to incorporate deoxycytidine as long as its concentration in the medium remains high enough. Measuring in vitro the phosphorylation of deoxycytidine by the pH 4.2 precipitate (which contained many kinds of enzymes) in crude extracts of the Thy A-11 and Thy A-35 mutants, with thymidine as the substrate, incorporation is proportional to the amount of extract added and to the incubation time. With deoxycytidine, 0.04 mg protein and 30 min incubation give the maximum incorporation values shown in Table 4. From the pH 4.2 precipitate of the extract in the Thy A-11 mutant, the produced dCMP is one-tenth the amount of dTMP. But in the Thy A-35 mutant, the amount of produced dCMP is about the same as that of dTMP. These data show that considerable deoxycytidine kinase or the enzyme which activates the phosphorylation of deoxycytidine is contained in the extracts of the Thy A-35 mutant.
DISCUSSION The intracellular dTTP pool exhibits two functions : one is a rate-limiting factor in the polymerization of DNA [5,6], and the other is a conversion to dTDPsugar by thymidine diphosphate glucose pyrophosphorylase for the biosynthesis of cell wall lipopolysaccharide [30]. Therefore in abnormal cell growth, the intracellular dTTP pool increases to a high value when the DNA elongation is inhibited by antibiotics (novobiocin and nalidixic acid) [24,31] or when the thimidine diphosphate glucose pyrophosphorylase is made defective by its gene mutation [5]. During the periods of thymine starvation in a thy- mutant ( E . coli K12T-U-Mp, 15T- and CR34), the dTTP
Deoxyribonucleoside Metabolism in E. coli C thy- ura- Mutant
172
Table 4. Phosphorylation of deoxyrihonucleosides by p H 4.2 precipitates in extructs of hoth Thy A-11 and Thy A-35 mutants Conditions were the same as those described in Fig.2. Enzyme assays were as described in Materials and Methods Bacterial strains
dNMP formed from
Deoxyribonucleoside added (dN) ~~-
dC
dT
nmol x min-' x mg protein-' ~~
Thy A-35
0.132
1.000
1.000
0.855
dC
~-
Deoxyribonucleoside
-
-
41.7
50.0
-
-
41.7
Deoxyribonucleoside monophosphate formed -~
~
dCMP
dU MP
nmol x min-' x mg protein-' Thy A-I1 Thy A-35
deoxycytidine deoxycytidine
1.000 1.000
dNMP
dNDP
dNTP
28.8 12.5 34.1 14.8
15.59 2.06 9.63 8.23
2.31 0.11 1.85 1.01
0.31 0.05 0.22 0.12
.
50.0
Table 5 Phoqhorylution and deaminution ofdeoxycytidine by p H 4 2 precipitatec in extracts of hoth T h j A-11 and Thy A-35 mutants Conditions were the same as those described in Table 4 Bacterial strdins
dN
nmol x mg protein-'
-~
Thy A-1 1
-
~~~~
~
dT
Sum of deoxyribonucleosides and deoxyribonucleotides formed after 30 min
1.046 0.561
pool decreases immediately to a very small value 1321, and in compensation the dCTP and dATP pools increase to very large values, but the dGTP pool was constant during incubation [23,31]. Also, the dTTP pool increases 1.5 - 6.0-fold compared to the original during uracil or cytidine starvation in the double pyrimidine-requiring mutant (uracil and cytidine auxotroph) [23]. These findings provide evidence that one pyrimidine nucleoside triphosphate (e.g. dCTP) pool accumulates during the inhibition of DNA synthesis while the other pyrimidine nucleoside triphosphate (e.g. dTTP) decreases, and vice versa. From these results, in the Thy A-35 mutant, the dTTP pool may increase to a large value as the dCTP pool decreases to a small value during exponential growth. This reduction of the dCTP pool causes a decreased rate of DNA replication. As a result, this reduced DNA replication reduces the DNA concentration (Table 1) as reported by Pritchard [4] and Pritchard and Zaritsky [3]. Although the rate of DNA synthesis in the dCyd-added culture increases twofold compared to that in its culture without addition (Fig.5), the dTTP pool in the dCyd-added culture does not decrease to one-half but increases to twice that of its culture without added dCyd. These data show that the DNA synthesis is absolutely dependent upon the dTTP pool.
As no rhamnose residue is detected in the core oligosaccharide of lipopolysaccharide of the E. coli C strain 1331, the dTDP-rhamnose 1341, which is a component of dTDP-sugar, would not be utilized for the lipopolysaccharide biosynthesis. From this, dTDPsugar pools in Thy A-1 1 and Thy A-35 mutants would show very high values during exponential growth. Comparing the data for the nucleotide pool in E. coli C and its mutant (Thy A-11 and Thy A-35) strains with those in other strains [23,24,28,29], in E. coli C and its mutant cells, pyrimidine ribonucleoside triphosphate (CTP and UTP) pools show much lower values than those of purine ribonucleoside triphosphate (ATP and GTP) pools, though the pyrimidine ribonucleoside triphosphate pools are of the same values as those of the purine ribonucleoside triphosphate pools in other strains (E. coli B and 15T-). As Thy A-11 and Thy A-35 mutants require uracil for growth, after the addition of uracil to the medium, the UTP pool in those mutants increases somewhat compared to the value of the UTP pool in E. coli C. In particular, the decrease of the UTP pool in the Thy A-35 mutant may give rise to the increase of other pyrimidine nucleotide (CTP, dCTP and dTTP) pools, but the addition of deoxycytidine without uridine and deoxyuridine increases the dTTP and dCTP pools, along with the rate of DNA replication during exponential growth. As the UTP pool does not increase by the addition of deoxycytidine, these results seem to point to mechanisms for the synthesis of pyrimidine nucleotide other than the decrease of the uridine nucleotide pool. Both lactobacilli [lo] and mammalian systems [l I ] can utilize deoxyribonucleosides for DNA synthesis, but E. coli cannot efficiently incorporate deoxyribonucleosides into DNA as these compounds are rapidly catabolized in the cells. Also, the mutant (OK 441) in the E. coli B which lacks four deoxyribonucleoside-catabolizing enzymes [ 161 did not incorporate any deoxyribonucleoside other than thymidine into its DNA, and only thymidine kinase
T. Ohkawa
but no other deoxyribonucleoside kinase could be detected in extracts of the mutant [15]. In Salmonella typhymurium, its cytidine (or deoxycytidine) deaminaseless mutant did not contain deoxycytidine kinase [23]. In the Thy A-35 mutant, DNA replication in the dCyd-added culture increases twofold compared to its rate in the culture without added dCyd, but the addition of other deoxyribonucleosides (deoxyadenosine, deoxyguanosine and deoxyuridine) than deoxycytidine and thymidine do not increase the rate of DNA replication. This result does not mean that the added deoxycytidine is first deaminated into deoxyuridine, after which DNA replication increases twofold. It is considered that the added deoxycytidine would be phosphorylated in the Thy A-35 mutant. As a result, Tables 3 and 4 show that the phosphorylation of deoxycytidine takes place in vivo and in vitro. These data indicate that extracts of the Thy A-35 mutant contain both thymidine kinase and considerable deoxycytidine kinase. or the enzyme which is able to phosphorylate deoxycytidine. It is concluded that the Thy A-35 mutant cells isolated from the E. coli C strain differ from the mutant cells from the E. coli B strain that were reported by Karlstrom [15]. As the activity of the deoxycytidine-catabolizing enzyme (deoxycytidine deaminase) is induced by the addition of deoxycytidine during exponential growth in E. coli [35], we must discuss the deamination of deoxycytidine added to the Media of Thy A-11 and Thy A-35 mutants. Deoxyuridine nucleotide pools show in vivo larger values ( 2 - 10-fold) than those of deoxycytidine nucleotide pools labeled with [3H]deoxycytidine during incubation for 45 min in these mutants. Therefore we performed the experiment to measure deoxycytidine nucleotide pools labeled with [3H]deoxycytidine in media containing excess deoxyuridine added in these mutants. Deoxyuridine nucleotide pools labeled with 3H show values one-half those of [3H]deoxycytidine nucleotide pools during incubation for 25 min (Table 3). Also, by the pH 4.2 precipitates purified partially in extracts of the Thy A-11 mutant, dUMP and dCMP are produced in the same amounts; but by the pH 4.2 precipitates in extracts of the Thy A-35 mutant, the amount of dUMP is only one-half that of dCMP (Table 5). These data present the possibility that considerable deoxycytidine kinase or the enzyme which is able to activate the phosphorylation of deoxycytidine could be purified from the extracts of the Thy A-35 mutant. As the deoxycytidine kinase has not been detected until now in E. coli B [15] and S. tjyhimurium [35], deoxycytidine may be phosphorylated by the enzyme which phosphorylates deoxyribonucleosides other than de-
173
oxycytidine in extracts of the Thy A-35 mutant. The purification of the enzyme and the phosphorylation of deoxycytidine in the Thy A-35 mutant are now under investigation. REFERENCES 1. Pritchard, R . H. & Zaritsky, A. (1970 Nature (Lond.) 226, 126 - 131. 2. Zaritsky, A. &Pritchard, R. H. (1971) J . Mol. Biol. 60, 65-74. 3. Zaritsky, A. & Pritchard, R. H. (1973) J . Bacteriol. 114, 824837. 4. Pritchard, R. H. (1974) Philos. Trans. R. Soc. L,ond. B, Biol. Sci. 267, 303 - 336. 5. Ohkawa, T. (1976) Eur. J . Biochem. 61, 81-91. 6. Beacham, I. R., Beacham, K., Zaritsky, A. Sr Pritchard, R. H. (1971) J . Mol. B i d . 60, 75-86. 7. Rachmeler, M., Gerhart, J. & Rosmerm, J . (1961). Biophys. Acta 49, 222 - 225. 8. Fangman, W. L. (1969) J . Bacteriol. 99, 681 -687. 9. Fangman, W. L. & Novick, A. (1966) J . Bacteriol. 91, 23902394. 10. Hoff-Jorgensen, E. (1952) Biochrm. J . 50,400-403. 11. Reichard, P. (1957) Acta Chem. Scand. 11, 11 -16. 12. Cohen, S. S. & Barner, H. D . (1957) J . B i d . Chem. 226, 631 64L. 13. Koch, A. L. & Vallee, G . (1959) J . Bid. Chem. 234, 12131228. 14. Manson, L. A. & Lampen, J. 0. (1951) J . B i d . Chem. 193, 539 - 549. 25. Karlstrom, H. 0. (1970) Eur. J . Biochem. 17, 68-71. 16. Karlstrom, H. 0. (1968) J . Buctrriol. 95, 1069-1077. 17. Stacev. K. A. & Simon. E. (1965)J. Bacteriol. 90. 554-555. 18. Burton, K. (1956) Biochem: J . 62, 315-323. 19. Lowry, 0. H., Rosebrough, N. J., F a n , A. L. & Randal, R. J. (1951) J . Mol. Biol. 193, 265-275. 20. Lomax, M. I. S. L Greenberg, G. R. (1967) J . B i d . Chem. 242, 109-113. 21. Roodman, S . T. & Greenberg, G. R. (1971) J . B i d . Chem. 246, 2699-2617. 22. Randerath, K. & Randerath, E. (1965) Anal. Biochem. 13, 575 - 579. 23. Neuhard, J. (1968) J . Bacteriol. 96, 1519-1527. 24. Fuchs, J . A. (1977) J . Bacteriol. 130, 957-959. 25. Bessman, M. J. (1963) Methods Enzymol. 6 , 166-176. 26. Okazaki, R. & Kornberg, A. (1964) J . B i d . Chem. 239, 269274. 27. Brachet, J. (1960) The Biological Role of Ribonucleic Acid, Elsevier, Amsterdam. 28. Filpula, D. & Fuchs, J. A. (1977) J . Bacteriol. 130, 107-113. 29. Manwaring, J . D. & Fuchs, J. A. (1977) J . Bacteriol. 130, 960 - 962. 30. Liideritz, O., Staub, A. M. & Westphal, 0. (1966) Bacteriol. Rev.30, 192-255. 31. Neuhard, J. & Thomassen, E. (1971) Eur, J . Biochem. 20, 36 - 43. 32. Ohkawa, T. (1975) Eur. J . Biochem. 60, 57-66. 33. Feige, U. & Stirm, S. (1976) Biochem. Biophys. Res. Commun. 71,566-573. 34. Okazaki, R., Okazaki, T., Strominger, Y. L. & Michelson, A. M. (1962) J . B i d . Chem. 237, 3014-3026. 35. O’Donovan, G. A. L Neuhard, J. (1970) Bacteriol. Rev. 34, 278 - 343.
T. Ohkawa, Department of Biochemistry, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa-shi, Ishikawa-ken, Japan 920
