Download Site-directed Mutagenesis of Arginine

Mol. Cells, Vol. 2, pp. 109-114
Site-directed Mutagenesis of Arginine-178 of Thymidylate Synthase
from Lactobacillus casei
Sung-Woo Cho*, and Soo Young ChoP
Department of Biochemistry, College of Medicine, University of Ulsan. Seoul 138-040, Korea;
IDepartment of Genetic Engineering. College of Natural Science, Hallym University, Chunchon
200-702, Korea
(Received on February 23, 1992)
X-ray structural studies have previously shown that Arg-178 of thymidylate synthase interacts with bound inorganic phosphate or with the S'-phosphate of the bound substrate
dUMP. The importance of Arg-178 to the structure/function of thymidylate synthase is
also indicated by its complete conservation among the 17 thymidylate synthases thus far
sequenced. In the present work, cassette mutagenesis has been performed for Arg-178 of
Lactobacillus casei thymidylate synthase. Eleven amino acid substitutions' have been obtained
for Arg-178. Methods have been developed for determination of functional and phenotypical characteristics for each of the newly synthesized mutant proteins. Functionally acceptable substitutions were defined by genetic complementation of thymidylate synthase deficient
cells and further characterized by determination of specific activity. Evaluation of kinetic
parameters of the mutants was performed in crude extract using S-fluoro-2'-deoxyuridylate
as an active site titrant Analysis of the mutants by genetic complementation indicates
that thymidylate synthase can tolerate a number of amino acid substitutions at that position
and shows that Arg-178 is not strictly required for catalytic activity.
is supported by the fact that this residue is completely
Thymidylate synthase (EC 2.1.1.4S) catalyzes conversion of dUMP and S,lO-methylenetetrahydrofolate
conserved in all TS' sequenced to date (Bzik et al.,
1987).
(CH 2H.Jolate) to dTMP and 7,8-dihydrofolate (H2folate). The catalytic mechanism of thymidylate synthase ·
In this report, we describe the strategy and preliminary results of an approach to understanding the st(IS) has been extensively studied (Santi and Danenructure of TS by saturation site-directed mutagenesis.
berg, 1984; Santi et al., 1987). The amino acid sequen"Replacement sets" were constructed in which Arg-178
ces of the enzyme from some 17 sources are known,
and the three-dimensional structures of TS from severesidue was replaced by a large number of substitural sources have been solved (Hardy et al., 1987). The
tions. The mutagenic DNA cassette contained a mixprimary sequences of TS have revealed that it is one
ture of 32 codons that encode 20 amino acids and
the amber (TAG) stop codon at the target site (Reiof the most conserved proteins known; the similarity
dhaar-Olson and Sauer, 1988). An advantage of synamong TS sequences implies a functional importance
for the conserved residues.
thetic TS gene as a mutagenesis vehicle (Climie and
Santi, 1990) is demonstrated by the construction of
The X-ray structures of the Lactobacillus casei TS-Pi
complex and the Escherichia coli TS' dUMp· lO-propaa series of 11 site-directed mutations at Arg-178 in
rgyl-S,8-dideazafolate ternary complex have provided
a single experiment The use of synthetic gene is furinsight about the possible roles of specific residues
ther enhanced by its high level ·of expression and by
of the protein, especially 4 conserved Arg residues
the ability to screen TS mutants by genetic comple(Arg-23, -178, -179, and -218 in L casel) which are
mentation in E. coli.
located within bonding distance of the liganded phosphate. Chemical modification and BC NMR studies
Materials and Methods
have also suggested that one or more arginine residues
are involved in binding of dUMP (Cipollo and DunMaterials
lap, 1979; Cipollo et al., 1982; Belfort et al., 1980). Two
Restriction endonucleases, polynucleotide kinase, T4
of these, Arg-178 and Arg-179, emanate from the other
DNA ligase, T4 DNA polymerase, and E. coli DNA
subunit of the homodimeric enzyme. Hence, it is reapolymerase I were purchased from New England Biosonable to predict that Arg-178 may playa ' role in
labs. [a-35S]dATP (>600 Ci/mmole) were purchased
binding the phosphate moiety of dUMP. The putative
from Amersham Corp. 'Sequenase DNA sequencing
importance of Arg-178 to the structure/function of TS
* To
whom correspondence should be addressed
The abbreviations used are: FdUMP, 5-fluoro-2'-deoxyuridylate; TS, thymidylate synthase.
© 1992 The Korean Society of Molecular Biology
Mutagenesis of Arg-178 in Thymidylate Synthase
110
kit was from U.S. Biochemicals. Oligonucleotides were
synthesized at the University of California, San Francisco, Biomolecular Resource Center. Other materials
have been reported (Bruice and Santi, 1982; Pinter
et al., 1988) or were commonly available.
Bacterial strains and plasmids
Strain DH5a [cj>80IacZLlM15; ara, ~(lac-proAB), rpsL
hsdR ; T. O. Baldwin, Texas A&M, College Station]
was used as the host strain for plasmid-mediated transformations for the initial isolation of mutants. The
Thy- E. coli strain X2913 (&hyA572), a gift from Dr.
Daniel V. Santi, University of California, San Francisco, USA, was used to test plasmids for TS activity
by genetic complementation and for the production
of recombinant TS. Strain JM101 [sup£, th4 ~(lac­
proAB) (F' , traD, proA +B+, lac[QZLlM15)] was used to
propagate M13 clones used for DNA sequencing. A
synthetic DNA (PSCTS9) was obtained from Dr. Daniel V. Santi, University of California, San Francisco,
USA
'
Plasmid construction
Mutations were performed by replacing the fragment with a synthetic DNA duplex (PSCTS9) that
presented mixed bases for the codons of interest. Since
the fragmeIit between two flanking sites (Clal and Mst
II) was too large (80 bp), pSCTS9/Smal, a derivative
of pSCTS9, was constructed by oligonucleotide-directed mutagenesis of synthetic TS gene cloned into M13
mpl9 vector (Fig. 1). The new construct denominated
as M13mpI9ffS/Smal is the same as the wild type,
except that Tyr-176 (TAC) is replaced by Gly (GGC)
to make a Smal site (CCCGGG) at His-174 (CAC),
r ._.
Hind In
£.coA I
_CI.
TS
RI
y--- D
• Ria Hili
Hln
PLATE
(IPTG + Xgal)
r"'aq....
(In..ft)
Stu. Plaq....
(NoInNfl)
~
7
dNTPs
O
)
Sja'
(Oligo)
O=~"' -~,
I
eamHI
• a5011
BomH I
,SS-DNA
~(TOmplOIO)
---====-.
sm, I SOli
b. '"." ...p . , I
Figure 1. Construction of pSCTS9/SmaI
9::
I
elmHI
• &5011
Mol. Cells
Pro-175 (CCG), and Gly-176 (GGC). Finally this
TS/Smal gene in Ml3mp19 vector was sub cloned into
pUCl8 to construct pSCTS9/SmaI plasmid. The modified DNA (PSCTS9/Smal) contains one additional
unique restriction site (Smal) between the two restriction sites (Cia I and MstII). Therefore, the flanking sites
used for Arg-178 mutation were Smal and MstII with
a length of 36 bp in pSCTS9/SmaI. General methods
for DNA manipulation were carried out as previously
described by Maniatis et al. (1982). DNA fragments
used for cloning were separated by electrophoresis on
0.8% low melting point agarose gels, and the excised
bands were used directly in the ligation reaction
(Crouse et aI., 1983).
Mutagenesis of Atg-178
A series of single amino acid substitutions of Arg178 were obtained by cassette mutagenesis of plasmid
pSCTS9/Smal (See Fig. 2). Plasmid DNA was digested
with Smal and MstII to remove a 36-bp fragment, and
vector DNA was purified by electrophoresis using 1%
low melting point agarose. The 36-bp Smal/MstII fragment was replaced with a 36-bp synthetic DNA duplex containing multiple substitutions on both DNA
strands at positions encoding Arg-178. The substitutions included a equal mixture of all four bases at
the first and second positions of the codon and an
equal mixture of G and C at the third position. Mutagenic oligonucleotides were annealed in 10-111 reaction
mixtures that co~tained 1, 5, 10, 50, and 100 pmoles
of each unphosphotylated oligonucleotide. The annealed DNA fragments were ligated with 0.5 Ilg of gelpurified vector DNA as described above and one half
of each ligation reaction was used to transform strain
DH5a to ampicillin resistance. The resulting colonies
were pooled by flooding the plates with 3 rnl of L
broth and collecting the cell suspension with a sterile
pipette. Plasmid DNA was prepared from the pooled
cells and used to transform E. coli strain X2913 (thy - ),
which was then plated on LB agar containing 100
Ilg/rnl of ampicillin and 50 Ilg/rnl of thymidine. Plasmid DNA was prepared from individual X2913 transformants and Arg-178 mutants were identified by dideoxynucleotide sequencing using plasmid DNA as
a template. Arg-178 mutants were further characterized
by their ability to grow on minimal agar in the absence of thymidine.
Protein preparations and enzyme assay
For small scale preparations, the transformed E. coli
strain X2913 (thy - ) was grown by inoculation of 50
rnl of LB containing 100 Ilg/rnl ampicillin and 50
Ilg/rnl thymidine with 0.25 rnl of overnight cultures
and incubation for 16-20 h at 37 °e, Cells were harvested by centrifugation washed with cold 150 mM NaCl,
then resuspended in 5 rnl of 100 mM Tris-HCl (PH
7.4), and 1 mM EDTA, and disrupted by sonication
on ice bath; cell debris was removed by centrifugation
at 10,000 X g for 15 min. Protein concentration was
determined by the method of Read and Northcote
(1981) using bovine serum albumin as a standard. TS
Sung-Woo Cho & Soo Young Choi
Vol. 2 (1992)
ABC
0
A
BCD
Restrict
B&C
111
zation of filters required approximately 24 h. Counting
efficiencies were determined using external standards.
Filtration efficiencies were routinely between 88 and
94% and were constant within one same experiment.
Results
~
t.W
Transformation
.,
32codons
20 amino acids
Sequence; screen/selecl for active
mutant in TS·deficient cells
Figure 2. Strategy for saturation site-directed mutagenesis of
synthetic TS gene and identification of catalytically. active
mutants. A fragment of the TS gene between two restriction
sites was removed and replaced by a mixture of oligonucleotides containing NNG/C at the Arg-178 codon. The resultant
plasmids were sequenced and screened in E. coli thy - cell
(X2913) for catalytic activity.
activity was assayed spectrophotometrically at 25 °C
using the conditions of Pogolotti et al. (1986). One
unit of activity is the amount of TS that catalyzes
the formation of 1 mmole of product per min. TS
synthesis was examined by SDS/PAGE of crude cell
extracts that were prepared by sonication of the overnight cultures grown in L broth containing 100 Ilg/rnl
of ampicillin and 50 Ilg/rnl of thymidine. SDS-PAGE
was performed by the method of Laemrnli (1970) and
gels were stained in Coomassie Brilliant Blue R-250.
Filter binding assay
The formation of the enzyme-CH)FdUMP complex
was performed in a standard assay mixture containing
50 mM TES (PH 7.4), 25 mM MgCh, 6.5 mM HCHO,
1 mM 2-mercaptoethanol, 0.l5 mM FAIL, (6- 3H)
FdUMP, and 10 Ilg of cell extract. The reaction was
initiated by addition of the cell extract in a total assay
volume of 100 j..ll. Mter 45-min incubation, 4O-j..ll aliquots were assayed in duplicate for complex formation
and 10 j..ll was removed for determination of CH)
FdUMP concentration in the assay mixture. Nitrocellulose membranes were soaked before use in 75 mM
potassium phosphate (PH 7.4). Filters which were not
wetted within 2 min were discarded. The filter discs
were placed on a filter manifold (Hoeffer-Scientific)
and a gentle vacuum was applied to remove excess
moisture. Without removing the vacuum, filters were
washed with 2 rnl of the same buffer and then 40
j..ll aliquots of the reaction mixture were applied to
the discs and allowed to permeate the membrane. Mter washing the filters with 6 rnl of the same buffer,
the damped filters were placed in scintillation vials
and dissolved in 10 ml of Aquasol. Optimal solubili-
Arginine-178 was chosen as a target for mutagenesis
because it is highly conserved and because it hasputative role in phosphate binding as revealed by previous solution and structural studies. The strategy used
in the construction and characterization of the replacement set is outlined in Figure 2. Following ligation
of oligonucleotide mixtures and initial transformation,
colonies were pooled, and the mutagenized plasmid
DNA pool was recovered and used to transform E.
coli X2913 (thy - ) to ampicillin resistance. Individual
mutants were identified by DNA sequencing, and the
resulting plasmids were used to re-transform X2913.
Plasmid DNA from the secondary X2913 transformants was again sequenced and tested for TS activity.
Passage of the mutagenized DNA mixtures through
several rounds of transformation ensured segregation
and repair of the heteroduplex DNA molecules that
were created during the construction of the mutants.
It was not practical to identifY all possible mutants
in a replacement set by this strategy, which would
require sequencing of over 160 clones to obtain a 95%
confidence level of obtaining a complete set. Generally, 20-30 isolates of a replacement set ,,:,ere seq~enced
to give 10-15 different mutants of a glVen reSIdue.
The results obtained with regard to complementation of X2913 for the different mutants of Arg-178
are shown in Table 1. Mter sequencing 30 clones,
we have isolated 11 of the 20 possible amino acid
substitutions, 1 amber codon, and three synonymous
Arg codons. The wild-type Arg codon, CGT, was not
found in the 30 plasmids sequenced, indicating the
mutagenesis efficiency was >97% in this experiment.
The 11 mutants arose at a frequency close to that
expected on the basis of the codon distribution in
the mutagenic DNA cassette (data not shown). Of the
11 different amino acid mutations obtained for Arg178, 3 mutants (Lys, Tyr and Thr) were able to complement the thyA deletion in X2913 on minimal agar
in the absence of thymidine, indicating that catalytically active TS was being synthesized. However, 8 mutants (Leu, Pro, Val, De, Ser, Asp, Glu, and Phe) did
not grow under the same conditions, suggesting that
the TS synthesized in these cells was incapable of
providing sufficient thymidylate to sustain growth. The
observation that at least three different residues can
be substituted at position 178 without complete loss
of activity allows us to conclude · that Arg-178 is not
strictly essential for TS function.
Analysis of crude cell extracts by SDS-PAGE showed that all 11 mutant plasmids encoding an amino
acid substitution at position 178 directed the synthesis
of a 37-kDa protein that comigrated with TS (Fig.
3). SDS-PAGE and activity measurements of crude
extracts prepared from each of the mutants expressed
112
Mutagenesis of Arg-I78 in Thymidylate Synthase
Mol. Cells
Table 1. Complementation screen and kinetic properties of Arg-I78 mutants
K.n
(~)
Amino acid
substitution
Genetic
complementation
Specific
activity
k"",
(sec ')
dUMP
Wild type
Lys-l78
Thr-178
Tyr-178
Active
Active
Active
Active
0.22
0.18
0.13
0.11
4.2
1.9
1.9
1.8
2.5
3 l.l
5.2
25
18
55
19
42
Leu-178
Ile-178
Pro-178
Phe-178
Val-I 78
Ser-178
Asp-I 78
Glu-178
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
<0.001
<0.001
<0.001
0.002
0.003
0.007
<0.001
<0.001
nda
nd
nd
nd
0.11
0.31
nd
nd
nd
nd
nd
nd
22 1.5
150.9
nd
nd
nd
nd
nd
nd
1253
996
nd
nd
CH 2H,Jolate
k""t!K,,,
dUMP (k ,)
(~ - ' sec - ')
CH 2 H,Jolate
1.68
0.06
0.23
0.07
0.23
0.03
0.11
0.04
nd
nd
nd
nd
0.0005
0.0021
nd
nd
nd
nd
nd
nd
0.0001
0.0003
nd
nd
and; not detectable.
AmWT K Y
T
L P
V
5
0
E
F
97 kDa--"
66 kDa--"
45 kDa--"
31 kDa--"
22 kDa--"
Figure 3. SDS-PAGE analysis of TS synthesis from X2913. Crude extracts from various Arg-I78 mutants; indicated by oneletter codes for the substituted amino acids except for the wild type (WT) and amber (Am). Lane I, molecular weight standard
proteins; lane 9, purified TS.
in X2913 showed that TS was expressed to a level
of -10% of the total soluble protein except the amber
substitution. Thus, the absence of catalytic activity is
not due to a lack of production or stability of the
different mutants.
Results on catalytically active enzymes were further
assessed by enzymatic assays of cell extracts. We have
determined that minimal detectable specific activity
in cell extracts is approximately I X 10- 4 U/mg protein, value 2,500 times lower than what has been observed for wild-type TS. Further characterization of functionally active TS presenting the different substitutions consisted in performing an estimation of k cat for
the different mutant TS'. Determination of k ca, involves
measurements of the actual amount of TS present in
cell extracts. This was performed by measuring the
formation of FdUMP-enzyme complex using the ftlter
binding assay. We have routinely measured the formation of complex at 3 different FdUMP concentrations; 76.3, 381.5, and 763 nM. At 76.3 nM, FdUMP
is below saturation and therefore provides values in
order to calculate ftltration efficiency for each mutant.
Calculations of the pmoles of dimer/mg cell protein
were performed assuming that the stoichiometry is 1.7
moles bound per mole of enzyme in the ternary complex. The k cat values for the native and mutant enzymes were calculated this way and are shown in Table
1.
As depicted in Figure 4, the interaction of substrates
with L casei TS is "Ordered Bi Bi", with dUMP binding fIrst (Danenberg and Danenberg, 1978). The pertinent kinetic expressions as derived by the method
of net rate constants (Cleland, 1975) are given in
Equations 1-4. Equation 1 gives the expression for
kcaJK~UMP at low CH2HJoiate concentration.
k cat
(1)
At saturating CH 2HJolate, Equation I reduces to
Equation 2.
k cat
(2)
Sung-Woo Cho & Soo Young Choi
Vol. 2 (1992)
k \ [dUMP]
TS .:;;;..;:::::::::=~ TS-dUMP
..
*2
,.1J " (C~"
TS-dUMP -
CHz
Ii. folate
,••• (
ks [= kcarl ..
Figure 4. Interaction of thymidylate synthase (fS) with substrates dUMP and CH 2HJoiate
Equation 3 gives the expression for kcaJK~H2H4folate at
low dUMP.
(3)
At saturating dUMP, Equation 3 reduces to Equation
4.
~+ks
(4)
In case of the active mutants, the Km values for
dUMP were increased about lO-12-fold for the Tyr
and Lys mutants, and about 2-fold for the Thr mutant.
The Km values for CH2HJoiate did not significantly
change in the Thr mutant, but was some 2-3-fold higher with the Tyr and Lys mutants. For inactive mutants (Val and Ser), the Km values icreased 6O-100-fold
for dUMP and SS-70-fold for CH2HJolate. The Km
values of the rest of the inactive mutants were too
low to measure. For all active mutants, kca\ values were
about 2 S-I, or 45% of the wild type. However, kca\
values of the inactive mutants were only 3 and 7%
of the wild type for Val and Ser, respectively. The
k cat values for mutations of Arg-178 were in all cases
lower than those observed with this method for the
wild type. Lys, Thr, and Tyr are optimal substitutions
in terms of k cat being only 2-3 times lower than for
the wild type.
Most interesting is the kcaJKm value. For dUMP,
the kcaJKm at saturating CH2HJoiate of the Thr mutant decreased 7-fold, whereas the Lys and Tyr mutants decreased 28- and 24-fold. For CH2HJolate, the
kcaJKm values at saturating dUMP for the Lys and
Tyr mutants decreased 6-8-fold, whereas for the Thr
mutant kcaJKm decreased only 2-fold. The kcaJKm values of the inactive mutants were undetectable or
much lower than those of the wild type.
Discussion
Our current understanding of the structure and catalytic activity of TS has been the result of biochemical study of many years. The Arg residues conserved
at positions 23, 178, 179, and 218 form a positively
charged binding surface for the 5' -phosphate of
dUMP. Five of the guanidineoNH groups are within
appropriate distance (2.5-3.2 A to provide hydrogen
bonding or electrostatic stabilization of the phosphate
anion of dUMP. Arg-178 is completely conserved in
113
all TS' sequenced to date (Bzilk et al., 1987), indicating
an important role in structure/function of the enzyme.
There are several features of the steady state rate
equations for TS which deserve emphasis. At saturating concentrations of CH 2HJolate, kcaJKm for dUMP
is a measure of the rate of association (k l ) of dUMP
with free enzyme (Eq.' 2); thus, Km values for dUMP
reflect kcaJkl . In contrast, at saturating dUMP concentrations kcaJKm values for CH2HJoiate are a combination of rate constants [k-Jcs/(k 4 + k s)J (Eq. 3 and 4)
and thus Km values of CH 2HJoiate can be considered
to be apparent binding constants. Since ok cat values
are similar for the active mutants and only 3-fold lower than the wild type, once substrates are bound within the ternary complex the catalytic steps of the reaction are only moderately affected by the nature of
the residue at 178.
The most revealing effect of the mutations is on
the net rate of asso~iation of dUMP with the enzyme
(k l); this represents a step of the reaction pathway
which can be readily measured as kcaJKm of dUMP
at saturating CH2HJoiate (Eq. 2). According to the
X-ray structure of the TS-Pi complex and molecular
modeling (Hardy et al., 1987), suggesting that a conformational change of the protein occurs to accommodate binding of dUMP, and pre-steady state kinetic
studies of the wild type enzyme (Mittelstaedt and
Schimerlik, 1986), the first rapid formation of a weak
complex (~~ 300 mM), is followed by a slower isomerization (k~ 300 S- I). The net rate constant calculated from these data (-1 ~ - IS- I) is in good agreement with the value of kl calculated from steady state
kinetics, suggesting that the same process is being
measured. It is proposed that the isomerization step
seen in pre-steady state kinetics reflects the anticipated
conformational change. The kl values obtained from
steady state kinetics must primarily reflect the slow
step of the interaction, and thus provide a kinetic
probe for the conformational change.
As shown in Table 1, kcaJKm of CH 2HJoiate at saturating dUMP is not very different in the Thr mutant.
Since this parameter measures [k-Jcs(k 4 + k s)J, the Thr
mutant does not significantly differ in any of the steps
after formation of the binary TS' dUMP complex. In
contrast, kcaJKm values of CH2HJoiate for the Lys and
Tyr mutants are reduced 6-8-fold compared to wild
type TS. Since kcat values of Tyr and Lys mutants
decreased only 2-fold compared to the wild type, and
the apparent Km of CH2HJoiate increased, the effects
can be attributed to binding of the cofactor. It is not
apparent why the active mutants would show the different effects, but it is unusual among those studied
in that it significantly affects both the rate of association of dUMP and binding of CH2HJolate. Although
the differential effects of the Arg-178 mutants have
allowed the aforementioned interpretations, it is apparent that although Arg-178 is bound to the phosphate
of dUMP, it is not essential for substrate binding or
catalytic activity. However it can be replaced by Lys,
Thr, and Tyr amino acids without drastic changes in
its biological activity.
0
114
Mutagenesis of Arg-178 in Thymidylate Synthase
How do we explain the enigma of the Arg-178
which is bound to the 5'-phosphate of dUMP, but
is not essential for activity? Our favored explanation
is that "plasticity" of the protein, and compensatory
function of other residues are important factors in the
retention of high activity of the 178 mutants. The guanidinium groups of Arg-23, Arg-179, and Arg-218 are
also bound to Pi (Hardy et al., 1987) or the 5' -phosphate of dUMP; it is proposed that upon mutation
of Arg-178 to a residue that cannot bind phosphate,
its important functions are transferred to the other
Arg residues. Studies on the mutagenesis of these
other Arg residues of TS are in progress in our laboratory.
A second enigma resides in the fact that Arg-178
is conserved in all TS' thus far sequenced (from bacteria, bacteriophage, protozoa, yeast, and mammals);
yet, it is not essential, or even significantly advantageous, for binding or catalysis. This feature has been
observed with other conserved residues which are also
apparently important to structure/function (Maley et
al., 1990; Dev et aI., 1989; Clirnie and Santi, 1990).
We surmise that conserved amino acid residues of
TS may serve crucial roles which are not revealed
from such studies.
The approach we have used has several practical
advantages. (1) A synthetic gene with conveniently
placed, unique restriction sites permits mutagenesis at
high mutagenesis efficiency. (2) A mutagenesis vector
which also serves as a high expression vector avoids
further subcloning. (3) A selection system for the desired property permits rapid identification of mutants
of interest. This combination of genetic and biochemical techniques could be used to address a broad range
of questions relating to the structure and function of
the enzyme. Adequate characterization of the corresponding mutant proteins would involve kinetic studies
of purified mutants.
Acknowledgments
We thank Dr. Daniel V. Santi for his generous gift
of synthetic TS gene (PSCTS9) and oligonucleotides.
This work was supported in part by grants from
Korea Science and Engineering Foundation (913-0401013-2) and the Asan Institute for Life Sciences to S.W. Cho (90-09-035).
Mol. Cells
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