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Journal of Bacteriology, September 1999, p. 5296-5302, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A- and T-Tract-Mediated Intrinsic Curvature in
Native DNA between the Binding Site of the Upstream Activator NtrC
and the nifLA Promoter of Klebsiella pneumoniae
Facilitates Transcription
Amrita Kaur
Cheema,
Nirupam
Roy
Choudhury, and
H. K.
Das*
Genetic Engineering Unit and Centre for
Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
Received 15 March 1999/Accepted 22 June 1999
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ABSTRACT |
The nif promoters of Klebsiella pneumoniae
must be activated by proteins bound to upstream sequences which are
thought to interact with the 
54-RNA polymerase
holoenzyme by DNA looping. NifA is the activator for most of the
promoters, and integration host factor (IHF) mediates the DNA looping.
While NtrC is the activator for the nifLA promoter, no IHF
appears to be involved. There are two A tracts and one T tract between
the upstream enhancer and the nifLA promoter. This DNA
segment exhibits anomalous electrophoretic mobility, suggesting
intrinsic sequence-induced curvature in the DNA. On the one hand,
mutation of the A tracts or T tract individually or together, or
deletion of the A tracts and the T tract reduces the anomaly; on the
other hand, creation of two additional A tracts enhances the anomaly.
Intrinsic curvature in the DNA has been confirmed by circular
permutation analysis after cloning the DNA fragment in the vector pBend
2 and also by electron microscopy. Computer simulation with the DNA
base sequence is also suggestive of intrinsic curvature. A
transcriptional fusion with the Escherichia coli lacZ gene
of the DNA fragment containing the nifLA promoter and the
wild-type or the mutated upstream sequences was constructed, and in
vivo transcription in K. pneumoniae and E. coli
was monitored. There was indeed very good correlation between the
extent of intrinsic curvature of the DNA and transcription from the
promoter, suggesting that DNA curvature due to the A tracts and the T
tract was necessary for transcription in vivo from the
nifLA promoter of K. pneumoniae.
| |
INTRODUCTION |
Biological reduction of atmospheric
dinitrogen is the exclusive preserve of a very limited number of
microorganisms. The molecular mechanism of the process has now been
worked out in considerable detail, and the gram-negative bacterium
Klebsiella pneumoniae has served as a model system for this
purpose. Twenty-one potential genes contained in eight operons have
been found in a single cluster (34). In addition to the
structural genes (nifHDK) for the enzyme nitrogenase, these
operons contain genes for cofactor synthesis, processing, carriers for
electron transport, and control elements (nifLA)
(34). The sigma factor involved in initiation of
transcription from the nif promoters is 54 or
N. The nif promoters are different from the
more common 70 promoters (2, 5, 23, 43) and
need the upstream activator NifA to initiate transcription (10,
37, 44). This is not a case of activation by recruitment of the
RNA polymerase holoenzyme to the promoter DNA (41). NifA is
counteracted by NifL in the presence of molecular oxygen and fixed
nitrogen (24, 42). The upstream bound NifA interacts with
the promoter-bound 54-RNA polymerase holoenzyme by DNA
looping mediated by the integration host factor (IHF) bound in between
the NifA binding site and the promoter (25, 46). It has been
observed in some in vitro experiments that the histone-like protein HU
can substitute for IHF at least partially (12, 39). On the
other hand, phosphorylated NtrC binds to two adjacent sites more than
100 bp upstream of the nifLA promoter and serves as the
activator of transcription (18, 21) by interacting with the
54-RNA polymerase holoenzyme bound to the promoter
(36). The actual activation process, of course, may be
dependent on oligomerization of NtrC involving protein-protein
interactions in addition to protein-DNA interactions (48).
It is, however, important to note that IHF does not bind to the
intervening sequence between the NtrC binding sites and the
nifLA promoter (45). The transcriptional activation by NtrC is, nevertheless, face-of-the-helix dependent (36), suggesting the involvement of DNA looping. The
mechanism for looping by which the activator and the RNA polymerase
holoenzyme would interact in this case thus appears to be different.
This assumes greater importance because the nifLA operon is
the master regulatory operon for all other nif operons.
The presence of intrinsic curvature in DNA because of the presence of
specific base sequences has been noticed, but most of these curvatures
are small compared to the marked effect produced by stretches of AT
tracts, each tract being about half a helical turn long
(29). Often a CA-TG doublet junction enhances the curvature
(4, 37).
Sequence-induced curved DNA is present in the replication origin of
bacteriophage lambda (49) and an autonomously replicating sequence of yeast (47). Curved DNA regions, inferred from
anomalous electrophoretic mobility, have also been found upstream of
the plasmid promoter PCIII, and their presence has been
found to increase in vivo and in vitro transcription, apparently
independent of any activator protein (40). On the other
hand, a significant percentage of Escherichia coli promoters
have A tracts in the immediate upstream region of the 
35 hexamers of
70 promoters, which have been predicted to confer a
sequence-induced curvature that is involved in transcription
(20). Interestingly, several in vitro studies using such
promoters have revealed that addition of RNA polymerase
70 holoenzyme alone was sufficient for transcriptional
activity and that no upstream activator was necessary (3, 26,
31), giving rise to the view that such bends could facilitate and
stabilize the initial binding of the RNA polymerase holoenzyme
(39). Sequence induced curvature in DNA has also been
inferred from computer analysis (6) of base sequence
upstream of the gln Ap2 promoter (11), but no
experimental data has been cited.
The evidence presented in this paper suggests that AT-tract-mediated
intrinsic curvature in native DNA is instrumental in ensuring the
interaction between the upstream activator and the promoter-bound RNA
polymerase-54 holoenzyme, resulting in transcription
from the promoter.
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MATERIALS AND METHODS |
Cloning of the nifLA promoter and the upstream
regulatory region of K. pneumoniae, and construction of the
deletion mutant.
The EcoRI-BglII (~1-kb)
fragment from pGPD44 (16), containing the K. pneumoniae nifLA promoter and the upstream activator site for NtrC
binding (bases 17681 to 18648 as described by Arnold et al.
[1]), was cloned at the
EcoRI-BamHI sites of pUC19. This was then
digested with HincII and SmaI, and the 553-bp
HincII-SmaI fragment (bases 17983 to 18536 as
described by Arnold et al. [1]) was cloned at the
HincII site of pUC19 in an orientation such that the
BamHI site in the pUC19 polylinker was upstream of the nifLA promoter and the HindIII site was
downstream (see Fig. 1). This construct was named pDJ22. The
BamHI-HindIII fragment from this construct
would be 583 bp long. To obtain the deletion derivative, the
HindIII-SalI fragment (571 bp) was removed
from pDJ22 and digested completely with Sau3A and the
largest fragment (275 bp), which had
HindIII-Sau3A ends, was collected after
electrophoresis in 2% agarose. The same
HindIII-SalI fragment was also partially digested with Sau3A, and the 213-bp
Sau3A-SalI fragment was isolated after similar
electrophoresis. These two fragments were then ligated and cloned in
the HindIII-SalI sites of pUC19. This
construct was named pDJ225.
Construction of other mutants.
The
BamHI-HindIII fragment from pDJ22 was cloned
in the phage vector M13mp19, and oligonucleotide-directed mutagenesis
was carried out as described by Kunkel (30) with the
Mutagene M13 kit (Bio-Rad). The oligonucleotides were synthesized in
the laboratory by the phosphoramidite method of solid-phase synthesis
(19), using a 391 DNA synthesizer (Applied Biosystems).
Acrylamide gel electrophoresis.
The DNA samples (~1 µg)
were electrophoresed in a 7.5% acrylamide gel with 25% glycerol and
1× TBE (88 mM Tris, 88 mM boric acid, 2.5 mM EDTA [pH 8.3]) at 4°C
for 36 to 40 h at 7 V/cm by using a vertical gel electrophoresis
apparatus (Hoefer).
Electron microscopy of DNA.
The plasmid containing the DNA
fragment to be analyzed was digested with restriction endonucleases and
electrophoresed on a 0.8% low-melting-temperature agarose (Sigma
Chemical Co.) gel. The gel piece(s) containing the DNA fragment
to be eluted was cut under long-wavelength UV light and kept in a
microcentrifuge tube. The tube was incubated at 65°C for 5 min, and
0.1 volume of 5 M NaCl was added to the molten agarose. It was mixed
well and incubated again at 65°C for another 5 min. The agarose was removed by extraction with an equal volume of phenol, (saturated with
100 mM Tris [pH 8.0]), and centrifugation in a microcentrifuge at
room temperature for 5 min. After the centrifugation, the upper, aqueous layer was transferred to a fresh microcentrifuge tube and
extracted with 2 volumes of diethyl ether. The two layers were allowed
to separate, and the upper layer was aspirated. Two volumes of ethanol
was then added to the aqueous layer and mixed well, and the DNA was
precipitated at 70°C for 30 min. The DNA was sedimented by
centrifugation in a microcentrifuge at 12,000 rpm (Eppendorf Plol
rotor) at 4°C for 15 min. The pellet was washed with chilled 70%
ethanol, dried in a vacuum desiccator, and dissolved in a buffer
containing 10 mM Tris and 1 mM EDTA (pH 8.0). DNA samples at
approximately 1 µg/ml were incubated with 10 mM ZnCl2 or
ZnSO4 (8, 22) at 55°C for 5 min and incubated
at 37°C for 5 min. The mixture was then brought to room temperature
before being prepared for electron microscopy.
Electron microscopy of the DNA fragment was carried out essentially as
described by Davis et al. (15). About 100 ng of DNA sample
was mixed with cytochrome c (from horse heart, type III [Sigma Chemical Co.]) to a final concentration of 0.005% and
formamide (EM Sciences) to a final concentration of 40% and spread on
water. The cytochrome c film containing DNA was gently
picked up on to a carbon-coated grid (400 mesh; Polysciences Inc.) and
stained with uranyl acetate (Polysciences Inc.) for 30 s (5 mM
stock in 50 mM HCl, freshly diluted 100-fold in 90% ethanol). The grid was then washed with 100% ethanol for 10 s. To increase the
contrast of DNA molecules further, the grid was rotary shadowed at an
angle of ~7° with Pt-Pd (80%:20%) (Polysciences Inc.). The grids
were then examined in a Philips EM 410 transmission electron
microscope. Two other methods of spreading have been checked. In the
method of Inman and Schnos (27) ~10-ng DNA samples (in 10 mM Tris-1 mM EDTA [pH 8.0]) were briefly mixed with the buffer
containing 65 mM Na2CO3, 10 mM EDTA, 33% HCHO,
and 40 mM HCl. Cytochrome c and formamide were then added to
the mixture (to final concentrations of 0.01 and 50%, respectively),
which was spread, stained, and rotatory shadowed. In the method
described by Chattoraj et al. (13), the carbon-coated grids
were treated with alcian blue (0.2% stock in 3% acetic acid, diluted
100-fold with water just before use) and then dried. About 5 ng of DNA
(in 10 mM Tris-1 mM EDTA [pH 8.0]) was applied directly onto the
grid and allowed to adsorb. The grid was washed in water, stained with
1% aqueous uranyl acetate solution, dried, and rotary shadowed with tungsten.
Construction of lacZ fusion and assay of
-galactosidase activity.
The low-copy-number broad-host-range
transcriptional fusion plasmid vector pGD499 (17) was used
as described previously (32). The DNA fragments containing
the mutated nifLA promoter and upstream sequence were
removed from M13mp19 as BamHI-HindIII fragments and cloned at the same sites of pGD499. E. coli
S17.1 or E. coli TB1 was transformed by the method of Cohen
et al. (14). The construct was mobilized from E. coli S17.1 into K. pneumoniae M5a1 by conjugation. A
-galactosidase assay (35) was performed under maximally
inductive conditions in the absence of NH4+ and
O2. E. coli cells were grown in M9 glucose
medium (devoid of NH4Cl) supplemented with Casamino Acids
(200 µg/ml). K. pneumoniae cells were grown in NFDM medium
(9), supplemented with Casamino Acids (500 µg/ml), in a
10-ml volume in acetylene reduction bottles (with rubber caps) sealed
with a metalic seal. These bottles were then flushed with argon for 5 min to remove traces of oxygen. The cultures were grown at 30°C until
the absorbance at 600 nm reached 0.3 to 0.5.
Mapping of the locus of bending.
The wild-type DNA
comprising nucleotides 131 to 38 with respect to the transcription
initiation site was amplified by PCR and cloned by blunt-end ligation
in the SmaI site of the plasmid vector pNEB 193 (construct
pAN 94). The DNA was then removed as an
EcoRI-BamHI fragment and cloned in the
XbaI site of the vector pBend 2 by blunt-end ligation after
end filling in (construct pADH I). This construct was then digested
separately with BamHI, SspI, NruI,
PvuII, EcoRV, XhoI, SpeI,
BglII, and MluI. The fragments released were then
electrophoresed in an acrylamide gel as described above.
| |
RESULTS |
The DNA stretch between the NtrC binding sites and the
nifLA promoter has anomalous electrophoretic mobility:
mutation or deletion of the two A tracts or one T tract present there
reduces the anomaly.
Figure 1
reveals the presence of two A tracts centered at positions 88 and
80 and one T tract centered at 67 with respect to the transcription
initiation site. The A tract at 80 is preceded by a GC at the 5' end.
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FIG. 1.
Sequence of the 583-bp
BamHI-HindIII fragment encompassing the
nifLA promoter and the upstream sequence of K. pneumoniae. The bases above the filled bars represent the two NtrC
binding sites (9). The bases above the open bars depict 26
and 12 regions containing the nifLA promoter
(5). The two A tracts and the T tract are between the NtrC
binding sites and the promoter.
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The 583-bp
BamHI-
HindIII fragment (Fig.
1)
migrated like a 690-bp fragment on acrylamide gel electrophoresis (Fig.
2; Table
1). Mutating an A or T tract in isolation
reduced the anomaly
in electrophoretic mobility to some extent (Fig.
2;
Table
1).
However, when two A tracts were mutated in conjunction or an
A
and a T tract were mutated together, reduction in the anomaly
in
electrophoretic mobility was substantial (Fig.
2; Table
1).
When the
83-bp stretch (positions
137 to
54) containing both
the A tracts
and the T tract was deleted, the resultant 500-bp
fragment migrated on
electrophoresis with practically no anomaly.
Mutation of bases
resulting in two additional A tracts at a 10-bp
interval upstream of
and in phase with the existing A tracts accentuated
the anomaly in
electrophoretic mobility.
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FIG. 2.
Electrophoretic mobility of the DNA fragments of the
wild-type and mutant nifLA promoters and the upstream
sequences obtained by digestion of the plasmids with
BamHI-HindIII (lanes c to j). Lanes: a and l,
HinfI-digested  ×174 replicative-form DNA. b and k,
HaeIII-digested pBR322 DNA; c, fragment with a deletion from
135 to 53 with respect to the transcription initiation site; d,
wild-type fragment; e, AAAA at positions 89 to 86 mutated to ACAC;
f, AAAAA at positions 82 to 78 mutated to ACACA; g, AAAA at
positions 89 to 86 mutated to ACAC and AAAAA at positions 82 to
78 mutated to ACACA; h, TTTT at positions 69 to 66 mutated to
TGTC; i, AAAA at positions 89 to 86 mutated to ACAC and TTTT at
positions 69 to 66 mutated to TGTC; j, CGGG at positions 99 to
96 mutated to AAAA and GCGG at positions 109 to 106 mutated to
AAAA.
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TABLE 1.
The apparent and relative sizes of the DNA fragments of
the wild type and various mutants with mutations in the upstream region
of the nifLA promoter as estimated from their respective
electrophoretic mobilitiesa
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|
Anomalous electrophoretic mobility on acrylamide gels has been accepted
as a manifestation of DNA fragments containing an
intrinsic
sequence-induced bend (
4,
33). We have therefore
inferred
that the two A tracts and the one T tract contribute
in a cumulative
manner to the curvature of the DNA stretch between
the NtrC binding
sites and the
nifLA promoter.
Predictions based on computer simulations of the DNA base sequence
are suggestive of the presence of intrinsically curved DNA.
Images
of DNA sequences of the wild-type and mutated DNA generated by using
CURVATURE (6) and NUVIEW (37) software are presented in Fig. 3. The estimate of
cumulative bending angle for the wild-type DNA was 79°, which was
reduced to 65° on mutation of the two A tracts. Deletion of the 83-bp
region containing the two A tracts and the one T tract resulted in a
cumulative bending angle of 50°, while introduction of two A tracts
in addition to the preexisting ones enhanced the cumulative bending
angle to 85°.
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FIG. 3.
Images of five DNA fragments (containing wild-type and
mutated nifLA promoter and upstream sequence) as predicted
by the CURVATURE (6) (a) and NUVIEW (37) (b)
software.
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Electron microscopy of the DNA fragments confirms the presence of
curvature in the DNA due to the A tracts and T tract.
The 583-bp
wild-type DNA fragment and the mutated versions were examined by
electron microscopy. Figure 4 shows
photographs of representative fields. The wild-type DNA and the DNA in
which two additional A tracts were introduced revealed many molecules with hairpin bends and some looped molecules. These curved structures very closely resemble the curved structures observed by Hoover et al.
(25) when they allowed IHF to bind upstream of the
nifH promoter of K. pneumoniae. The fragment in
which the two A tracts were mutated and also the one in which both the
A tracts and the T tract were deleted showed predominantly linear
molecules and no molecule with a hairpin bend or looped molecule at
all. We have screened for molecules which exhibited bend angles of
>90°, and the results are presented in Table
2.
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FIG. 4.
Representative electron micrographs of the fragments
containing the nifLA promoter and the upstream sequence. (A)
Wild type (583 bp). (B) Two existing A tracts have been mutated (583 bp). (C) An 83-bp stretch containing the two A tracts and one T tract
has been deleted (500 bp).
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TABLE 2.
Data showing the population of bent
moleculesa as determined from the electron
micrographs of various DNA samplesb
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The frequency of bent molecules has been found to be similar
irrespective of the method of spreading
used.
Experimental mapping of the locus of bending.
The wild-type
DNA comprising the region from 131 to 38 with respect to the
transcription initiation site was cloned in the vector pBend 2 (28), and the electrophoretic mobilities of DNA fragments
generated from the duplicated circularly permuted restriction sites
were determined (Fig. 5). An analysis of
the mobilities is presented in Table 3. A
plot of the relative sizes of the fragments against the base position
enabled us to map the locus of bending to be around the G residue at
position 95 with respect to the transcription start site (Fig.
6).
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FIG. 5.
Electrophoretic mobility of DNA fragments generated by
digestion of pADH.1 (a construct containing a 94-bp stretch comprising
the nifLA promoter and the upstream region cloned in the
plasmid vector pBend 2). Lanes: a and m, HaeIII-digested
×174 RF DNA; b and l, HinfI-digested ×174 DNA; c to
k, the fragment released on digestion of pADH 1 with BamHI
(lane c) with SspI (lane d) (there is an additional
SspI site in pBend 2 besides the two in the polylinker; the
upper band in lane d is a result of this); with NruI (lane
e), with PvuII (lane f), with EcoRV (lane g),
with XhoI (lane h), with SpeI (lane i), with
BglII (lane j), and with MluI (lane k).
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TABLE 3.
Apparent and the relative sizes of the DNA
fragmentsa as estimated from their respective
electrophoretic mobilitiesb
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FIG. 6.
Permutation analysis of the 258-bp fragment generated by
digestion of pADH 1 with different restriction endonucleases containing
the putative bent locus in the upstream region of the nifLA
promoter of K. pneumoniae.
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Mutations that modulate DNA bending also modulate transcription
from the nifLA promoter in vivo.
Transcriptional
fusions with lacZ were constructed by using the
low-copy-number plasmid pGD 499 (17), and -galactosidase activity was determined in K. pneumoniae M5a1 and E. coli TB1 (Table 4). Mutation of the
individual A tracts or T tract caused a small but significant reduction
in transcription from the nifLA promoter, but mutation of
both the A tracts or one A tract and the T tract or deletion of 83 bp
(positions 137 to 54) containing both the A tracts and the T tract
indeed resulted in substantial reduction. Mutation of bases at 10-bp
intervals upstream of but in phase with the existing A tracts, creating
two additional A tracts, led to considerable stimulation in
transcription from the nifLA promoter. Mutation of the 10 bases at positions 131 to 122, keeping the same purine-pyrimidine
bias, did not have any deleterious effect on transcription.
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TABLE 4.
-Galactosidase activities of lac fusions of
the region containing the nifLA promoter and
upstream sequencea
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| |
DISCUSSION |
The presence of intrinsic curvature in DNA due to the presence of
two A tracts and one T tract between the NtrC binding sites and the
nifLA promoter of K. pneumoniae appears certain
on the basis of anomalous electrophoretic mobility and electron
microscopy (Fig. 2, 4, and 6; Table 1). Mutation in an individual A
tract or T tract or two tracts simultaneously or deletion of all the three tracts reduces both curvature in DNA and in vivo transcription from the promoter, and the magnitude of reduction of both phenomena is
in the same order, as mentioned above (Tables 1 and 4). This is the
first report on the parallelism between A- and T-tract-mediated bending
of native DNA and transcription in vivo. However, synthetic curved DNA
sequences have been shown to be capable of effectively replacing DNA
fragments containing a CRP binding site upstream of the gal
promoter of E. coli (7). We have assumed that the role of the A- and T-tract-mediated bent DNA is to facilitate the
interaction of RNA polymerase-54 holoenzyme with the
upstream activator NtrC, which results in transcription from the
nifLA promoter of K. pneumoniae. This is analogous to the situation with the NifA-dependent promoters, although
in the latter cases IHF is instrumental in promoting the interaction
(25, 46).
It has been reported that open-complex formation in vitro at the
glnAp2 promoter of E. coli present on linear DNA
was dependent on the native sequence of nucleotides situated in the
intervening region between the enhancer and the promoter
(12). Replacement of the native sequence of nucleotides by
random sequence affected open-complex formation. Computer simulation
based on the Trifonov algorithm (6) led Carmona et al. to
infer the presence of an intrinsic bend in the DNA between the enhancer
and the glnAp2 promoter. No experimental data has been
presented to substantiate bending.
However, the interesting observation was that the replacement of the
native sequence of nucleotides, which possibly induced the bend in the
DNA, did not affect open-complex formation in vitro when the promoter
was present on supercoiled DNA. Plasmid DNA in vivo is more likely to
be supercoiled than linear or open circular. Therefore, if the in vitro
experiments can be extrapolated to the in vivo situation, the putative
bent DNA upstream of the glnAp2 promoter is inconsequential
in vivo.
Carmona et al. (12) have also inferred the presence of bent
DNA upstream of the K. pneumoniae nifLA promoter by computer simulation, but again no physical evidence has been provided by actual
experimentation. Our experiments have established that bent DNA is
essential for optimal expression in vivo, at least for the
nifLA promoter.
| |
ACKNOWLEDGMENTS |
We are grateful to E. N. Trifonov and M. Bansal for making
available computer softwares for analysis of curvature in DNA and to S. Adhya for providing pBend 2. Long and useful discussions with M. Bansal
are also gratefully acknowledged. D. J. Sengupta and A. Manna
deserve thanks for helping with some of the experiments.
The work was supported by the Department of Biotechnology, Government
of India.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Genetic
Engineering Unit, Jawaharlal Nehru University, New Delhi 110067, India. Phone: 91(011)610-1044.. Fax: 91(011)616-5886. E-mail:
hirendas{at}hotmail.com.
| |
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Journal of Bacteriology, September 1999, p. 5296-5302, Vol. 181, No. 17
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
