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Molecular and Cellular Biology, November 2000, p. 8001-8007, Vol. 20, No. 21
University of Texas Medical Branch, Sealy
Center for Molecular Science, Galveston, Texas 77555-1061
Received 1 August 2000/Returned for modification 11 August
2000/Accepted 16 August 2000
O6-Methylguanine (m6G) is formed by the
action of alkylating agents such as
N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and
it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human
DNA polymerase O6-Methylguanine
(m6G) is formed in DNA by treatment with alkylating agents such as
N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG). m6G is highly mutagenic, and the mutagenic and carcinogenic
potency of alkylating agents closely parallels their ability to form
m6G in DNA (31). In yeast as well as higher eukaryotes, m6G
specifically induces G · C to A · T transition mutations
(30, 35).
Alkylation at the O6 position of guanine has a profound
effect on base pairing properties. Melting studies of DNA duplexes containing m6G have shown that the m6G · T base pair is
energetically less stable than the m6G · C base pair
(10), and nuclear magnetic resonance studies have indicated
that the m6G · T base pair is less hydrogen bonded than the
m6G · C base pair (33, 34). Nevertheless, DNA
polymerases incorporate T opposite m6G more often than C, because the
m6G · T mispair retains the Watson-Crick geometry more closely
than the m6G · C base pair. At neutral pH, C is inserted
opposite m6G via a wobble configuration, but the phosphodiester links
both 3' and 5' to the C are distorted in this base pair (18, 19,
24, 40, 41, 46).
m6G is a block to synthesis by prokaryotic and eukaryotic DNA
polymerases. Extensive steady-state kinetic analyses have indicated that the Escherichia coli Klenow fragment is inhibited by
m6G both at the step of insertion of a nucleotide opposite the lesion and at the step of extension from the m6G · C or m6G · T
base pair (7). Sequenase (T7 DNA polymerase) is partially
inhibited by m6G at both these steps (42). Eukaryotic DNA
polymerase The Saccharomyces cerevisiae RAD30 gene functions in
error-free replication of UV-damaged DNA, and RAD30-encoded
polymerase Steady-state kinetic studies with yeast and human Pol Here, we show that yeast and human Pol DNA substrates.
The m6G-containing 75-nucleotide (nt)
template oligomer was synthesized by Midland Certified Reagent Co.
(Midland, Tex.). DNA substrates S-1, S-2, S-3G, S-3A, S-3T, and S-3C
were generated by annealing the 75-nt template,
5'-AGCTACCATGCCTGCCTCAAGAATTCGTAAm6GATGCCTACACTGGAGTACCGGAGCATCGTCGTGACTGGGAAAAC-3', which contained an m6G at the underlined position 45 nt
from the 3' end, to the 32-, 44-, and four different 45-nt 5'
32P-labeled oligomer primers: N4456
(5'-GTTTTCCCAGTCACGACGATGCTCCGGTACTC-3'), N4309
(5'-GTTTTCCCAGTCACGACGATGCTCCGGTACTCCAGTGTAGGCAT-3'), or oligonucleotides that contain N4309 with one additional G, A, T, or C
residue at its 3' end, respectively. In the control undamaged DNA
substrates, the 75-nt template with the undamaged G residue at position
45 was used. The sequences of DNA substrates containing 18-nt template
oligonucleotides annealed to 12-nt primer DNA are shown in the figures.
The sequence of the 18-nt oligonucleotides, used as markers, was
5'-AGAGGAAAGTAGXGAAGG, which
contained a C (C marker), an A (A marker), a T (T marker), or a G (G
marker) residue at the underlined X position.
DNA polymerase reactions.
Yeast and human Pol Steady-state kinetic analyses.
Analysis of kinetic
parameters for deoxynucleotide incorporation opposite m6G or primer
extension from this lesion was done as described before (3, 12,
29). Briefly, yeast Pol MNNG sensitivity and mutagenesis in yeast.
All the yeast
strains used for these experiments were derived from EMY74.7. For
determining MNNG-induced forward mutations at the
CAN1S locus, cells were grown overnight in yeast
extract-peptone-dextrose (YPD) medium, sonicated to disperse clumps,
washed, and resuspended in 0.1 M sodium acetate buffer, pH 5.0. Appropriate volumes of stock MNNG solution (made as 1 mg/ml in 0.1 M
sodium acetate buffer, pH 5.0, and stored at Genetic evidence for the involvement of DNA polymerases
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Replication past
O6-Methylguanine by Yeast and Human DNA
Polymerase
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(Pol) in the replicative bypass of m6G lesions in
DNA. The formation of MNNG-induced mutations is almost abolished in the
rad30
pol32 double mutant of yeast, which lacks the
RAD30 gene that encodes Pol and the Pol32 subunit of DNA
polymerase 
(Pol). Although Pol can function in the mutagenic
bypass of m6G lesions, our biochemical studies indicate that Pol is
much more efficient in replicating through m6G than Pol. Both Pol
and Pol insert a C or a T residue opposite from m6G; Pol,
however, is more accurate, as it inserts a C about twice as frequently
as Pol. Alkylating agents are used in the treatment of malignant
tumors, including lymphomas, brain tumors, melanomas, and
gastrointestinal carcinomas, and the clinical effectiveness of these
agents derives at least in part from their ability to form m6G in DNA.
Inactivation of Pol could afford a useful strategy for enhancing the
effectiveness of these agents in cancer chemotherapy.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, required for lagging strand DNA synthesis, is strongly
blocked one base before m6G, indicating an inhibition of nucleotide
insertion opposite the lesion (42). m6G also blocks DNA
polymerase 
, which is involved in base excision repair
(38). Thus, although the m6G · T base pair is more
Watson-Crick-like in geometry than the m6G · C pair, DNA
polymerases are quite inefficient in incorporating even a T opposite m6G.
(Pol) replicates past a cis-syn
thymine-thymine (T-T) dimer by inserting two adenines across from the
two thymines of the dimer (16, 44). In humans, a defect in
the yeast RAD30 counterpart causes the variant form of
xeroderma pigmentosum (XP-V) (15, 27), and because of a
deficit in error-free replication of UV-damaged DNA, XP-V cells are
hypermutable with UV light. As a consequence, XP-V individuals suffer
from a high incidence of sunlight-induced skin cancers.
have shown
that this enzyme replicates through the T-T dimer with the same
efficiency and fidelity as through the equivalent undamaged Ts
(17, 44). Both yeast and human Pol are low fidelity
enzymes, misincorporating nucleotides with a frequency of
10
2 to 103 (17, 45). We have
previously suggested that Pol has a flexible active site which
renders the enzyme more tolerant of DNA distortions, enabling it to
synthesize DNA past a T-T dimer (17, 44, 45).
replicate through the m6G
lesion by inserting a C or a T residue opposite the lesion. Although
our genetic studies in yeast indicate a role for both Pol and Pol
in the replicative bypass of m6G lesions in DNA, our biochemical
studies provide evidence that Pol is much more efficient at it than
Pol. We discuss the possibility that inactivation of Pol could be
useful for enhancing the effectiveness of alkylating agents in cancer chemotherapy.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
were
purified as described previously (16, 17). Standard DNA
polymerase reactions (10 µl) contained 40 mM Tris-HCl (pH 7.5), 5 mM
MgCl2, 1 mM dithiothreitol, bovine serum albumin (100 µg/ml), 10% glycerol, 100 µM deoxynucleoside triphosphate (dNTP),
and 20 nM 5' 32P-labeled oligonucleotide primer annealed to
an oligonucleotide template. Reactions were initiated by adding a DNA
polymerase enzyme, yeast Pol (10 nM), yeast Pol (2.5 nM), or
human Pol (2.5 or 5 nM). For the identification of the nucleotide
incorporated opposite m6G, we used an 18-nt template primed with a
12-nt oligomer. Pol and Pol bind poorly to this short DNA
substrate; therefore, higher amounts of Pol (40nM) and Pol (10 nM) were used in these experiments. After incubation for 5 min at
30°C, reactions were terminated by the addition of 40 µl of loading
buffer containing 20 mM EDTA, 95% formamide, 0.3% bromphenol blue,
and 0.3% cyanol blue. The reaction products were resolved on 10 or
20% polyacrylamide gels containing 8 M urea and were dried before
autoradiography at 
70°C with intensifying screens. Gel band
intensities were quantified by PhosphorImager and the ImageQuant
software (Molecular Dynamics).
was incubated with increasing
concentrations of a single deoxynucleotide (0 to 2,400 µM) for 1 min
under standard reaction conditions. Gel band intensities of the
substrates and products were quantified by PhosphorImager and the
ImageQuant software. The percentage of primer extended was plotted as a
function of dNTP concentration, and the data were fitted by nonlinear
regression using SigmaPlot 5.0 to the Michaelis-Menten equation
describing a hyperbola, v = (Vmax × [dNTP]/(Km + [dNTP]). Apparent
Km and Vmax steady-state parameters were obtained from the fit and were used to calculate the
frequency of deoxynucleotide incorporation
(finc) and extension (f0ext) using the following
equation: finc or ext = (Vmax/Km)incorrect
pair/(Vmax/Km)correct
pair.
20°C) were added to
1-ml suspensions of cells adjusted to 1.5 × 108 cells
per ml. Samples were incubated in the presence of MNNG with vigorous
shaking for 20 min at 30°C. The reaction was terminated by the
addition of 1 ml of 10% sodium thiosulfate. Appropriate dilutions of
cells were plated on YPD for viability determinations and on synthetic
complete medium lacking arginine but containing canavanine for
determining the frequency of can1r mutations.
Plates were incubated at 30°C and counted after 3 and 4 to 5 days for
viability and mutagenesis determinations, respectively.
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
and in the mutagenic bypass of m6G lesions in yeast.
To identify the
DNA polymerase(s) involved in replication past m6G in eukaryotes, we
examined, in S. cerevisiae, the frequency of MNNG-induced
CAN1s to can1r forward
mutations in deletion mutants of the POL32 gene that encodes
one of the subunits of the replicative DNA polymerase Pol
(11) and of RAD30 that encodes Pol
(16). Yeast Pol is comprised of three subunits of 125, 58, and 55 kDa. The 125-kDa catalytic subunit and the 58-kDa subunits
are essential for viability, but the 55-kDa subunit, which is encoded
by the POL32 gene, is not essential (11).
mutant compared with the wild type but was not
affected in the rad30 mutant. MNNG-induced
can1r mutagenesis was, however, almost abolished
in the pol32 rad30 double mutant. These results
indicate a role for Pol and Pol in error-prone replication of m6G
in DNA, and they further suggest that Pol performs this task in a
more error-prone manner than Pol. Consistent with the absence of
m6G-induced mutagenesis in the rad30 pol32 strain,
this strain exhibits enhanced sensitivity even at the low MNNG
concentrations used in these experiments (Fig. 1B).
View larger version (15K):
[in a new window]
FIG. 1.
MNNG-induced mutations at the CAN1 locus in
rad30 and pol32 yeast strains.
can1r mutation frequency (A) and viability (B)
in MNNG-treated yeast strains are shown. Cells grown overnight in YPD
medium were treated with MNNG at the concentrations indicated for a
20-min period. Appropriate dilutions were spread onto synthetic
complete medium lacking arginine and containing canavanine for the
determination of CAN1s to
can1r mutation frequencies and onto YPD plates
for viability determinations. Each curve represents the average of two
or more experiments. 
, EMY74.7 (wild type) (RAD30 POL32);
, YPO-69 (pol32); 
, YR30.2 (rad30);
, YR30.97 (rad30 pol32).
Efficient m6G bypass by yeast Pol.
To examine the ability
of yeast Pol and Pol to replicate across m6G in DNA, we performed
running-start and standing-start experiments using a 75-nt template DNA
substrate containing a single m6G lesion 45 nt from the 3' end and
primed with a 5' 32P-labeled 32- or 44-nt oligomer,
respectively. Under conditions where approximately 50% of the primers
were extended by both DNA polymerases (Fig.
2A), yeast Pol replicated through only
~7% of the m6G lesions (Fig. 2A, lanes 2 and 4) compared to
synthesis on template containing an undamaged G residue (Fig. 2A, lanes 1 and 3). In contrast, compared to replication on undamaged DNA (Fig.
2A, lanes 5 and 7), yeast Pol replicated through m6G ~10 times
more efficiently (~70%) than yeast Pol (Fig. 2A, lanes 6 and 8).
Furthermore, yeast Pol exhibits a strong stall site right before the
lesion, indicating an inhibition of insertion across from m6G, and
another weaker stall site opposite the lesion, indicating some
inhibition of extension 3' to the modified base (Fig. 2A, lane 2).
These two stall sites are also observed with yeast Pol, but they are
much weaker (Fig. 2A, lane 6). These results indicate that whereas the
m6G lesion presents a strong block for yeast Pol, yeast Pol
bypasses this lesion quite readily.
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and yeast Pol on an 18-nt template containing a G or an
m6G residue at position 13 from the 3' end and primed with a
standing-start 12-nt primer (Fig. 2B). The relative electrophoretic mobilities of the products of the DNA synthesis reaction were compared
to 18-nt oligomer markers containing a C, an A, a T, or a G residue at
position 13, on a 20% polyacrylamide gel (Fig. 2B, lanes 1 to 4). As
expected, DNA synthesis on undamaged templates by yeast Pol or yeast
Pol resulted in the incorporation of the correct C residue across
from G at position 13 (Fig. 2B, lanes 5 and 7). On the damaged
template, yeast Pol replicated through the lesion by inserting a C
(~30%) or a T (~70%) across from m6G (Fig. 2B, lane 6), while
yeast Pol replicated through this lesion by inserting a C (~60%)
or a T (~40%) (Fig. 2B, lane 8). Thus, while both DNA polymerases
replicate through m6G in an error-free as well as an error-prone
manner, yeast Pol inserts the correct residue C about twice as
frequently as yeast Pol.
Steady-state kinetic analyses of base insertion and extension
during m6G bypass by yeast Pol.
To characterize further the
bypass of m6G lesion by yeast Pol, we measured the kinetic
parameters of base insertion and extension during translesion DNA
synthesis. The kinetics of insertion of a single deoxynucleotide
opposite an m6G and the kinetics of addition of the next correct
nucleotide to various 3'-primer termini situated across from m6G were
determined as a function of deoxynucleotide concentration under
steady-state conditions (3, 12, 29). From the kinetics of
deoxynucleotide incorporation, the steady-state apparent
Km and Vmax values for
each deoxynucleotide were obtained from the curve fitted to the
Michaelis-Menten equation. The frequency of nucleotide
misincorporation, finc, and the frequency of
mismatch extension, foext, were
calculated as the ratio of the efficiency
(Vmax/Km) of incorrect
nucleotide incorporated or extended from, to the efficiency
(Vmax/Km) of correct
nucleotide incorporated or extended from, respectively (Tables
1 and 2).
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incorporates C opposite the m6G lesion about 20-fold less
efficiently than C opposite G (Table 1), and extension from the C
· m6G base pair is about twofold less efficient than extension from
the C · G base pair (Table 2). Yeast Pol incorporates T
opposite m6G about sevenfold better than T opposite G (Table 1), and it
extends the T · m6G base pair about 40-fold more efficiently
than the T · G mispair (Table 2). The order and the ratio of
deoxynucleotide insertion opposite m6G by Pol were T:C:A:G and
~23:15:2:1 (Table 1), and the order and the frequency of extension
from different 3'-terminal deoxynucleotides paired with the m6G
template residue were C:T:G:A and ~64:26:2:1 (Table 2). Thus,
opposite m6G, yeast Pol inserts the incorrect T slightly better than
the correct C, but it is more efficient at extending from C opposite
m6G than from T opposite this lesion. From these analyses, we estimate that yeast Pol would bypass m6G by inserting a C or a T residue and
then extending from the resulting base pair with an efficiency of
2.0 × 102 and 1.3 × 102,
respectively, relative to the efficiency of insertion of a C opposite
an undamaged G residue and extension from this base pair (Tables 1 and
2). These kinetic results are in accord with the level of incorporation
of C (60%) and T (40%) during replication through an m6G lesion by
yeast Pol (Fig. 2B).
m6G bypass by human Pol.
We also examined the ability of
human Pol (hPol) to bypass the m6G lesion. As shown in Fig.
3A, hPol replicated through m6G
~70% as efficiently as through undamaged DNA. hPol exhibits two
stall sites, one right before m6G and the other opposite the lesion,
indicating that there is some inhibition of deoxynucleotide insertion
opposite m6G as well as inhibition of extension from the base opposite
the lesion. hPol bypasses m6G by inserting a C or a T opposite this
lesion about equally frequently (Fig. 3B, lane 6).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our genetic studies in yeast indicate that Pol and Pol
provide alternate pathways for the mutagenic bypass of m6G. Since the
frequency of MNNG-induced can1r mutations is
reduced in the pol32 mutant but not in the
rad30 mutant, these studies further suggest that Pol
bypasses the m6G lesion in a more error-free manner than Pol. Also,
our biochemical studies indicate that Pol replicates through m6G
more accurately than Pol, as it inserts a C opposite this lesion
about twice as frequently as Pol. Although Pol replicates through
the m6G lesion quite inefficiently in vitro, association with RFC and PCNA may enhance this reaction in vivo. Because of its required role in
DNA replication, Pol will be the first polymerase to arrive at the
m6G lesion; however, at times Pol may stall at the lesion site,
necessitating the participation of Pol in this process.
Rad6-Rad18-dependent protein ubiquitination (1) may play a
crucial role in the replacement of Pol by Pol.
Yeast and human Pol replicate through a cis-syn T-T dimer
with the same efficiency and accuracy as undamaged DNA (17,
44). m6G is, however, somewhat inhibitory to Pol, and as
indicated from steady-state kinetic studies, replication through this
lesion is about 50-fold less efficient than the replication of
undamaged G (Tables 1 and 2). These observations may be reflective of the more frequent formation of a T-T dimer than an m6G lesion in DNA,
and that could have imposed a more intense selection pressure on Pol
for the more efficient and accurate bypass of a T-T dimer than the m6G lesion.
Kinetic studies with the Klenow fragment of E. coli DNA
polymerase I have indicated that relative to the insertion of a C opposite G, this enzyme incorporates a C or a T residue opposite m6G
poorly, with a frequency of 1.3 × 104 and 3.3 × 104, respectively (7). Also, relative to
the extension from a G · C base pair, the Klenow fragment
extends from an m6G · C or an m6G · T base pair with a
frequency of 4.3 × 103 and 12.9 × 103, respectively (7). By contrast, Pol
inserts a C or a T residue opposite m6G with a frequency of 4.3 × 102 and 6.8 × 102, respectively
(Table 1), and extends from the m6G · C or the m6G · T
base pair with a frequency of 4.7 × 101 and
1.9 × 101, respectively (Table 2). Thus, Pol is
over 100-fold more efficient than the Klenow fragment in inserting a C
or T opposite from m6G, and it is also more efficient in extending from
the resulting base pair.
In eukaryotes, replicative DNA polymerases Pol (42) and,
as shown here, Pol are inhibited by m6G. Although Pol can
replicate through m6G in DNA, it does so 10,000-fold less efficiently
than the replication of undamaged DNA, and Pol inserts primarily a T
residue (~95%) opposite m6G (38). Further, our genetic
studies in yeast have yielded no evidence that might impute a role for Pol in m6G bypass. Thus, Pol is unlikely to have a role in m6G bypass.
Pol differs from other eukaryotic DNA polymerases in its ability to
replicate through the cis-syn T-T dimer and 8-oxoguanine (8-oxoG) lesions efficiently and accurately (13, 17, 44). We
show here that Pol bypasses the m6G lesion with a reasonable efficiency, and it is more adept at inserting the correct nucleotide C
opposite m6G than Pol. All of these lesions distort the DNA helix.
Although the two T's in the T-T dimer can base pair with A's, a dimer
is still a block to most DNA polymerases, presumably because of the
intolerance of their active site for the DNA distortion caused by the
dimer (2, 14, 21, 22, 43). 8-oxoG in the syn
conformation mimics T and has the correct geometry to form a stable
base pair with A via two hydrogen bonds, whereas 8-oxoG in the
anticonformation forms a normal Watson-Crick base pair with C that
involves the same three hydrogen bonds as in the G · C base pair
(23, 25, 28, 32). In the 8-oxoG · C base pair,
however, the template strand is distorted significantly in the vicinity
of the lesion (23, 25, 28, 32). Eukaryotic replicative DNA
polymerases , , and 
bypass 8-oxoG by incorporating an A
rather than a C opposite the lesion (13, 36), presumably because their active site is unable to adapt to the distortion conferred by the 8-oxoG · C base pair. Pol, on the other
hand, bypasses 8-oxoG by predominantly inserting a C opposite the
lesion (13). Pol is also more efficient at inserting a C
opposite the m6G lesion than Pol, even though the phosphodiester
backbone is distorted in the m6G · C base pair (18, 19, 24,
40, 41, 46). The m6G · C base pair, however, is more
hydrogen bonded than the m6G · T base pair (33, 34).
The ability of Pol to replicate through the T-T dimer, the 8-oxoG
lesion, and the m6G lesion could derive from an active site which is
indifferent to DNA distortion caused by these lesions but which can
utilize the ability of these modified bases to form base pairs.
The involvement of Pol in m6G bypass suggests that inactivation of
this enzyme could be useful for increasing the effectiveness of
alkylating agents in cancer treatment. Chloroethylating agents, in
combination with methylating agents such as procarbazine and temozolomide, are presently used to treat malignant tumors,
particularly lymphomas, brain neoplasms, malignant melanomas, multiple
myeloma, and gastrointestinal carcinomas (6). The clinical
effectiveness of these agents is attributed, in part, to their forming
O6-alkylguanine adducts in DNA (26).
Intrinsic and acquired resistance to alkylating agents, however, limits
the efficacy of these drugs, and
O6-methylguanine DNA methyltransferase (MGMT),
which transfers the methyl group from m6G to its active site cysteine
residue, contributes to this resistance (4, 39). High levels
of MGMT prevent the cytotoxic effect by removing
O6-alkylguanine DNA adducts, and inactivation of
MGMT by a potent inhibitor, O6-benzylguanine,
sensitizes cells to killing by temozolomide (6). Consistent
with the involvement of Pol in m6G bypass, XP-V cells exhibit
enhanced sensitivity to alkylating agents that form
O6-alkylguanine in DNA (37). Thus, in
cells where MGMT has been specifically inactivated by
O6-benzylguanine, additional inactivation of
Pol by a specific inhibitor may confer enhanced sensitivity to
alkylating agents, arising from a defect in both the removal of m6G and
its bypass during replication. Hence, simultaneous inactivation of MGMT
and Pol may prove to be an effective strategy for enhancing the
sensitivity of tumor cells to alkylating agents and for augmenting the
effectiveness of these drugs in chemotherapy.
In humans, DNA mismatch repair (MMR) potentiates the cytotoxicity of
O6-alkylguanine, and cells acquire resistance to
these agents by inactivating MMR (5, 20). As Pol inserts
a C or a T residue opposite m6G, and since the human Msh2-Msh6 protein
complex binds the m6G · T and m6G · C base pairs equally
well, removal of either of these nucleotides (C or T) from the newly
synthesized DNA strand by the MMR system might lead to a reiterative
process of excision and synthesis, resulting in cell death
(8). This could account for the role of MMR in enhancing the
cytotoxicity of alkylating agents. In MMR-deficient cells, even the
inactivation of MGMT fails to sensitize cells to temozolomide
(9), and thus, in the absence of MMR, even high levels of
O6-alkylguanine adducts are not cytotoxic. In
cells inactivated for Pol, however, sensitivity to alkylating agents
may be maintained even in the absence of functional MMR.
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ACKNOWLEDGMENTS |
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We thank P. M. Burgers for yeast Pol.
This work was supported by NIH grants GM19261 and CA80882.
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FOOTNOTES |
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* Corresponding author. Mailing address: University of Texas Medical Branch, Sealy Center for Molecular Science, 6.104 Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061. Phone: (409) 747-8601. Fax: (409) 747-8608. E-mail: lprakash{at}scms.utmb.edu.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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