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(Received for publication, October 23, 1995; and in revised form, January 10, 1996) From the
Integration of the human immunodeficiency virus (HIV) DNA into
the host genome is an obligatory process in the replicative life cycle
of the virus. This event is mediated in vitro by integrase, a
viral protein which binds to specific sequences located on both
extremities of the DNA long terminal repeats (LTRs). These sites are
highly conserved in all HIV genomes and thus provide potential targets
for the selective inhibition of integration. The integrase-binding site
located on the HIV-1 U5 LTR end contains two adjacent purine tracts on
opposite strands, 5` . . . GGAAAATCTCT-3`/3`-CCTTTTAGAGA . . . 5`, in
parallel orientations. A single strand oligonucleotide
5`-GGTTTTTGTGT-3` was designed to associate with these tracts via its
ability to form a continuous alternate strand DNA triplex. Under
neutral pH and physiological temperature, the oligonucleotide, tagged
with an intercalator chromophore oxazolopyridocarbazole, formed a
stable triplex with the target DNA. The occurrence of this unusual
triplex was demonstrated by both DNase I footprinting and electron
microscopy. The triplex inhibits the two steps of the
integrase-mediated reactions, namely, the endonucleolytic cleavage of
the dinucleotide 5`-GT-3` from the 3` end of the integration substrate
and the integration of the substrate into the heterologous target DNA.
The midpoints for both inhibition reactions were observed at
oligonucleotide concentrations of 50-100 nM. We believe
that these results open new possibilities for the specific targeting of
viral DNA LTR ends with the view of inhibiting integration under
physiological conditions.
The integration of the human immunodeficiency virus (HIV) ( The present work shows that a
11-mer oligonucleotide-intercalator conjugate (OPC linked to the 5`-end
of 5`-GGTTTTTGTGT-3`) (i) readily forms a stable triplex with a DNA
fragment containing the U5 LTR end sequence at neutral pH and
physiological temperature and (ii) selectively inhibits the
IN-catalyzed integration of the U5 LTR end into heterologous DNA in
vitro. The formation of this unusual triplex is demonstrated by
both footprinting assay and electron microscopy.
Formation of a triple helix by oligonucleotides in a
sequence-specific manner is limited to polypurine tracts of duplex DNA.
Recent theoretical and experimental work has demonstrated the ability
of oligonucleotides to bind to oligopurine sequences which alternate on
the strands of duplex
DNA(15, 20, 21, 22, 31, 32, 33, 34, 35, 36, 37) .
Three different types of alternate triple helix-forming
oligonucleotides have been described: (i) two pyrimidine
oligonucleotides can be linked either by their 3` or by their 5` ends
to allow the recognition of alternating polypurine
sequences(31, 32) ; (ii) a purine oligonucleotide that
binds in antiparallel orientation can be linked to a pyrimidine
oligonucleotide which binds in an opposite orientation with respect to
the oligopurine
target(33, 34, 35, 36, 37) ;
and (iii) a single (T/G)-containing oligonucleotide can interact with
two oligopurine tracts that alternate on both strands of the target
DNA. Indeed, (G/T)-containing oligonucleotides may adopt either a
parallel or an antiparallel orientation with respect to the oligopurine
target depending upon the sequence considered and, in particular, the
number of 5`-GpT-3` and 5`-TpG-3` steps present in the
sequence(38) . We adopted the third strategy and synthesized an
11-mer oligonucleotide 5`-GGTTTTTGTGT-3` designed in order to create a
stable alternate strand DNA triplex with the 5`-GGAAAATCTCT-3` motif
located at the extremity of the HIV-1 U5 LTR (Fig. 1). It was
assumed that this oligonucleotide will form two mini triple helices,
the first one involves antiparallel binding of the third strand to the
5`-AGAGA-3` motif (referred as antiparallel domain), and the second
involves parallel binding to the 5`-GGAAAA-3` motif (referred as
parallel domain) of the LTR extremity (Fig. 1B).
Despite the fact that the parallel mini helix contains GGC triplets in
a noncanonical orientation, the orientation of the third strand was
chosen to be consistent with empirical rules for design of the
(G/T)-containing third strand(38) . According to these rules,
two parameters were taken into consideration for the stability of these
mini helices: (i) there is only one GpT step in the 5`-GGTTTT motif and
(ii) more than 50% of the putative mini triple helix is composed of TAT
triplets. Initial results indicated that the association of the
oligonucleotide alone with the DNA target resulted in the formation of
a triple helix. The complex, however, was poorly stable even after an
overnight incubation at 4 °C. In order to increase the stability of
the triple helix, the oligonucleotide was conjugated with the
intercalating chromophore OPC. The resulting compound, termed HIVS-OPC,
is shown in Fig. 1A. As a control, an alternative
5`-GGTTTTGGTTT-3` oligonucleotide was also conjugated to OPC (termed
HIVT-OPC, Fig. 1A) to be used for its inability to form
a stable triplex with the U5 LTR.
Figure 1:
Structure of triple helix-forming
oligonucleotides conjugated with OPC and schematic presentation of the
triplex. A, HIVS-OPC is a oligonucleotide conjugate whose
binding oligopurine-oligopyrimidine sequence is located in the U5 LTR
HIV-1 end region. HIVT-OPC was synthesized to serve as a control
conjugate. B, schematic representation of the alternate strand
DNA triplex formed between HIVS-OPC and the target
sequence.
The ability of the HIVS-OPC
conjugate to bind to double-stranded DNA via the formation of a triple
helix has been initially examined by gel-retardation experiments (data
not shown). Results from a 21-mer LTR U5 substrate provided evidence
for the formation of a stable complex between the partners. These were
confirmed by DNase I footprinting using a labeled 42-mer target at
different HIVS-OPC concentrations (Fig. 2). The footprinting
experiments were performed on the two strands of the DNA target. These
were involved in the formation of either antiparallel or parallel
triplets. The results are presented on Fig. 2, A and B, respectively. The antiparallel domain which interacts with
the 3`-end of HIVS-OPC was fully protected for concentrations of
HIVS-OPC above 100 nM. The protection spanned over the entire
length of the target sequence and extended to the parallel domain (Fig. 2A). Simultaneously, the second strand was itself
also protected over the whole length of both domains in the same range
of the concentrations of HIVS-OPC (Fig. 2B). Control
experiments were performed with conjugate HIVT-OPC. This
oligonucleotide contained the intact parallel binding domain but was
scrambled within the antiparallel binding domain (see Fig. 1) so
that two mismatched triplets would be formed at positions 7 and 10
(from the 5` end) upon its binding to the target. Actually no
protection was observed even at micromolar concentrations (data not
shown). Taken together, these results provided strong evidence for the
necessity of simultaneous binding of the (G/T)-containing
oligonucleotide-OPC conjugate to both target domains in order to give
rise to a stable triple-helix complex. It is worthy to note that
(G/T)-containing conjugate HIVS-OPC was not optimized for the junction
step. In particular, the oligonucleotidic moiety was not deleted at the
junction to accommodate crossing of the major groove as suggested by
Beal and Dervan(33) . This alteration appears therefore
dispensable in the context of (G/T) oligonucleotides. However, we
cannot rule out that a punctual deletion at the junction may improve
the overall stability of the triple helical complex.
Figure 2:
DNase I footprinting of a 42-base pair
fragment in the presence of HIVS-OPC. A, protection of the
oligonucleotide containing 5`-AGAGATTTT-3` site as a function of
HIVS-OPC concentration. B, protection of the complementary
strand as a function of HIVS-OPC concentration. Lanes 1, 2, and 3, G, (G + A), and T ladders,
respectively; lanes 4-9, decreasing concentrations of
HIVS-OPC from 2 µM to 10 nM, respectively; lane 10, control of digestion; lane 11, the target
fragment only.
In parallel, we
used electron microscopy to detect and localize the binding of the
HIVS-OPC conjugate on its target DNA sequence. Previously, this
technique has been applied successfully to detect triplexes formed
between DNA and either biotinylated TFO or peptide nucleic acid
oligomers with streptavidin used as a label; triplexes are visualized
readily as streptavidin beads on DNA
molecules(27, 39, 40) . The electron
micrographs presented in Fig. 3were obtained with the
3`-biotinylated HIVS-OPC conjugate and plasmid DNA containing a cloned
U5 copy of the terminal sequences. They confirm the formation of a
highly specific and selective complex, located 263 bp from the nearest
end of DNA. This coincides well within experimental error (21 bp) with
the position of the target sequence 273-283 bp from the same end
of DNA molecule.
Figure 3:
Electron microscopy visualization of the
triplex. The sites of triplex formation are seen as beads corresponding
to the streptavidin molecules (see C). A, micrographs
of the complexes bio-3`-GGTTTTTGTGT-5`-OPC-pU5/NheI plasmid
DNA-streptavidin. The arrows indicate the streptavidin
molecules. Micrographs were taken in a annular dark-field mode on
rotary-shadowed molecules. B, histogram of the distribution of
bound streptavidin on 49 DNA molecules. The position of the peak center
is 263 bp from the nearest end. The arrow indicates the
position of the U5 LTR target site. C, schematic illustrating
our approach.
To demonstrate that the oligonucleotide-OPC
conjugate could inhibit the processing and integration of the U5 LTR,
we applied a quantitative assay as described previously(17) .
The assay involves synthetic double-stranded oligonucleotide which
match one of the HIV-1 LTR extremities as the strand transfer substrate
and a heterologous plasmid DNA as a target substrate. It concerns the
first step of integration, namely, endonucleolytic cleavage, where
integrase removes the dinucleotide 5`-GT-3` from the 3` end of LTR U5 (Fig. 4A, lane 9). Increasing the HIVS-OPC
concentration from 10 nM to 2 µM in the reaction
mixture resulted in a notable inhibition of the endonucleolytic
cleavage (Fig. 4A, lanes 1-8). In
contrast, the control oligonucleotide HIVT-OPC, even at high
concentrations, had no effect on this activity (Fig. 4B). As expected, the LTR U3 substrate was
normally processed by integrase (Fig. 5A, lane
9) in the presence of either HIVS-OPC or HIVT-OPC, thus reflecting
the absence of triplex formation with this segment of DNA (Fig. 5, A and B, respectively).
Figure 4:
Effect of oligonucleotide conjugates
HIVS-OPC and HIVT-OPC on the IN-mediated nucleolytic cleavage of LTR U5
substrate under the standard conditions (see ``Materials and
Methods''). The reaction products were electrophoresed in a 18%
denaturing polyacrylamide gel. The positions of the labeled strand of
DNA substrates (21-mer) and the nucleolytic cleavage products (19-mer) are indicated. A, LTR U5 cleavage was tested
in the presence of decreasing concentrations of HIVS-OPC. Lanes
1-8, decreasing concentrations of HIVS-OPC from 2 µM to 10 nM, respectively; lane 9, cleavage without
HIVS-OPC. B, the same assay was performed using LTR U5
cleavage in the presence of decreasing concentrations of HIVT-OPC; lane 10, control without HIVT-OPC and without
integrase.
Figure 5:
Effect of oligonucleotide conjugates
HIVS-OPC and HIVT-OPC on the IN-mediated nucleolytic cleavage of LTR U3
substrate. The positions of the longer strand transfer
(autointegration) are indicated. A, LTR U3 cleavage was tested
in the presence of decreasing concentrations of HIVS-OPC. Lanes
1-8, decreasing concentrations of HIVS-OPC from 2 µM to 10 nM, respectively; lane 9, cleavage without
HIVS-OPC. B, the same assay was performed using LTR U3
cleavage in the presence of decreasing concentrations of HIVT-OPC; lane 10, control without HIVT-OPC and without
integrase.
To further
evaluate the influence of HIVS-OPC and HIVT-OPC on integration, the LTR
U5-GT and LTR U3-GT oligonucleotides were used as substrates and the
pSP65 vector as a target DNA. In the absence of the TFO conjugate,
integrase yielded an integration of about 20% for U5-GT and of
5-10% for U3-GT, consistent with a previous report(41) .
Adding HIVS-OPC into the integration mixture resulted in a strong
inhibition of the reaction with a midpoint of integration efficiency
corresponding to 60 nM HIVS-OPC (Fig. 6A).
Noteworthy, the midpoint of inhibition for the endonucleolytic cleavage
occurred at the same range of HIVS-OPC concentrations (Fig. 4A, lanes 5-8). With the control
HIVT-OPC, the integration processed normally at any concentration
tested (Fig. 6B). As expected, neither HIVS-OPC nor
HIVT-OPC had a visible effect on the integration of LTR U3-GT into
plasmid DNA (Fig. 7).
Figure 6:
Specific inhibition of LTR U5-GT
integration in the presence of oligonucleotides conjugates HIVS-OPC and
HIVT-OPC. A, specific inhibition of LTR U5-GT integration in
the presence of decreasing concentrations of HIVS-OPC. Lanes
1-6, decreasing concentrations of HIVS-OPC from 2 µM to 50 nM, respectively; lane 7, control without
HIVS-OPC and without integrase; lane 8, control without
HIVS-OPC. B, the same assay was performed using LTR U5-GT
integration in the presence of decreasing concentrations of
HIVT-OPC.
Figure 7:
Lack of the effect of the oligonucleotide
conjugates HIVS-OPC and HIVT-OPC on LTR U3-GT integration. A,
LTR U3-GT integration in the presence of decreasing concentrations of
HIVS-OPC. Lanes 1-6, decreasing concentrations of
HIVS-OPC from 2 µM to 50 nM, respectively; lane 7, control without HIVS-OPC and without integrase; lane 8, control without HIVS-OPC. B, the same assay
was performed using LTR U3-GT integration in the presence of decreasing
concentrations of HIVT-OPC.
The results presented here clearly
demonstrate through two independent methods the occurrence of an
alternate strand DNA triplex near the integrase-binding site of the U5
LTR HIV-1 end. Our DNase I footprinting experiments show that binding
of the designed oligonucleotide to a 42-mer target sequence results in
the formation of a triplex that is stable at neutral pH and
physiological temperature. Electron micrographic data, obtained with
the same target DNA cloned within a plasmid DNA, confirm the high
selectivity of the triplex formation as an uniquely positioned complex
over the whole sequence of plasmid DNA. The inhibitory effect of the
triplex formation was proven through the inhibition of the two HIV-1
integrase-mediated reactions, namely, the endonucleolytic cleavage of
the substrate and its subsequent integration into the heterologous DNA.
This inhibition was observed at relatively low concentrations of
HIVS-OPC (50-100 nM) probably due to the beneficial
influence of the 5`-end-conjugated intercalator chromophore. Together with our previous findings on the in vitro inhibition of U3 LTR HIV-1 end integration via a canonical
triplex, this work extends of the range of possible targets on HIV DNA.
It may also constitute a new basis for a pharmacological strategy
against the propagation of AIDS.
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10359-10364
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)genome into the host genome is mediated by the viral
protein integrase (IN)(1) . After the reverse transcription of
the HIV genomic RNA, two reactions occur, catalyzed by the viral IN
enzyme, a site-specific removal of two nucleotides (5`-GT-3`) from the
3` ends of the long terminal repeats (LTR) of the viral DNA and the
integration of the recessed viral DNA into the host
genome(2, 3) . Efficient methods have been reported to
investigate integrase activity by analyzing the in vitro reaction products(4) , and several families of compounds
that inhibit integrase activity have now been
identified(5, 6, 7, 8, 9, 10, 11, 12, 13) .
However, none of them displayed a strong and/or selective inhibitory
effect. Our approach toward obtaining more potent inhibitors involves
the targeting of the LTR extremities that contain the cis-acting sequences required for a correct integration,
further shown to be binding sites for integrase(14) . These
sequences can be considered as a potential target for the selective
inhibition of integration by double-stranded DNA binding ligands.
Depending on the sequence context, selective recognition of
double-stranded DNA can be chiefly achieved either with minor
groove-binding oligopeptides or with triple helix-forming
oligonucleotides(15) . For instance, we previously demonstrated
that the minor groove binder netropsin selectively binds to an (A
+ T)-rich sequence located in the MMLV LTR end and consequently
inhibits the in vitro integration process(9) .
Furthermore, a derivative of netropsin was capable of blocking early
steps of MMLV replication in vivo, most likely by interfering
with integration of proviral DNA(16) . Recently, we have
extended this approach to HIV. The HIV U3 LTR end contains a short
purine-pyrimidine which could be selectively targeted by a purine 7-mer
triple helix-forming oligonucleotide coupled to the intercalating
chromophore oxazolopyridocarbazole (OPC)(10, 17) .
However, theoretical considerations indicate that 7-mer
oligonucleotides are only poorly selective, thus preventing the use of
too short TFOs for in vivo experiments(18, 19) . To overcome this
limitation, it was necessary to increase the length of the target
sequence. We noticed that the IN-binding site located near the U5 LTR
HIV-1 end contains two adjacent purine tracts oriented in parallel on
opposite DNA strands, i.e. 5`-GGAAAATCTCT-3`/3`CCTTTTAGAGA-5`.
Theoretical (20) and experimental data (21, 22) have pointed out that TFO with natural
5`-3` phosphodiester bonds can recognize the two alternate purine
tracts simultaneously by switching from one strand to the other and
thus providing alternating triplexes of opposite polarities, parallel
and antiparallel ones(20) .
Synthesis of TFO-OPC
Oligonucleotides were
synthesized on Applied Biosystems model 381A DNA synthesizer. For
modified oligonucleotides, the 5` linker was obtained with the amino
link 2 phosphoramidite base. The OPC-derived oligonucleotides were
prepared essentially as described by Gautier et
al.(23) . Briefly, peroxidase was added to a solution of
the oligonucleotide linker and 2-methyl-9-hydroxyellipticinium acetate
in 50 mM phosphate buffer, pH 7.4, in the presence of 20
mM hydrogen peroxide(24) . Conjugates were purified by
denaturing gel electrophoresis, visualized by UV shadowing and direct
fluorescence of the OPC. The oligos were desalted by chromatography on
the Sephadex G-10 phase. The concencentration of conjugates was
determined spectrophotometrically.Footprinting Experiments
A 0.1-µg portion of
the 42-mer oligonucleotide
5`-AGAATTAGCCCTTCCAGTACTGCTAGAGATTTTCCACACGAT-3` was labeled by
polynucleotide kinase and annealed to the complementary strand in 40
µl in order to obtain target DNA. DNase I footprinting was
performed in a buffer containing 20 mM Tris-HCl, pH 7.0, 5
mM MgCl
, 2 mM MnCl, 0.5
mM spermine, target DNA (10 nM), and additional
nonspecific unlabeled DNA (60 ng). In the standard assay, TFO-OPC was
added to the reaction mixture at different concentrations (see figure
legends), and the mixture was incubated at 30 °C for 30 min.
Digestion was started by addition of DNase I (3 units/ml) and stopped
after 2 min by adding EDTA (10 mM), sodium acetate (0.3 M), and carrier tRNA (5 µg). Products of the reaction were
subsequently precipitated with ethanol, dried, and resuspended in
formamide/EDTA gel-loading buffer. The cleavage products were loaded on
18% denaturing gel and visualized by autoradiography.Oligonucleotide Sequencing
Modified Maxam-Gilbert
sequencing reactions were used to generate G, (G + A), and T
ladders of end-labeled DNA(25) . For G reactions, 9 µl of
end-labeled DNA in TE buffer were incubated for 10 min at room
temperature with 1 µl of 1/100 dimethyl sulfate in water. For the
(G + A) reaction, 9 µl of labeled DNA in TE buffer (10 mM Tris, 1 mM EDTA) were mixed with 1 µl of 1 M piperidine formate (pH 2.0) and incubated for 5 min at 65 °C.
For T reactions, 9 µl of labeled DNA were heated at 90 °C for 2
min, cooled quickly to room temperature, mixed with 1 µl of 3
mM KMnO
, and incubated further for 7 min at room
temperature. The reaction was finally quenched with 1 µl of allyl
alcohol. Then all three mixtures were treated for 15 min with 1 M pyrrolidine at 90 °C, dried, and resuspended in formamide/EDTA
gel-loading buffer.HIV-1 LTRs Integration Reaction
Double-stranded
oligonucleotides were used as HIV-1 DNA substrates for the integration
assay. Sequences (21-mer) corresponding to the U3 and U5 LTR ends were
5`-GAGTGAATTAGCCCTTCCAGT-3` and 5`-GTGTGGAAAATCTCTAGCAGT-3`,
respectively. They were 5`-labeled by polynucleotide kinase and
annealed to their unlabeled complementary strands thus giving the
desired substrates (called LTR U3 and LTR U5, respectively). For the
integration reactions, LTR U3-GT and LTR U5-GT (LTR U3 and LTR U5
lacking the terminal dinucleotides 5`-GT-3`, respectively) were
used as substrates. HIV-1 IN was expressed in Escherichia coli BL21. The bacterial strain carrying the expression vector was
kindly provided by Dr. Craigie (National Institutes of Health,
Bethesda, MD), and purified as described previously(26) .
Protein purity was checked by SDS-polyacrylamide gel electrophoresis
and the concentration was determined spectrophotometrically. HIV-1 LTR
integration was quantified as described previously(26) .
Briefly, standard assay medium for the integration into the pSP65
vector composed of 20 mM HEPES, pH 7.0, 10 mM MnCl, 10 mM dithiothreitol, 10% glycerol, 0.1
mg/ml bovine serum albumin (integration buffer), and 15 ng of pSP65
plasmid DNA. TFO-OPC was first incubated for 30 min at 30 °C with
LTR U3-GT or LTR U5-GT substrate (10 nM) under the same
conditions used for the reaction of footprinting. 1 µl of this
mixture was then added to 4 µl of the integration buffer, and the
reaction was started by the addition of 5 pmol of HIV-1 IN. Incubation
was continued for 40 min at 30 °C. The reaction was stopped by
adding 10 mM EDTA, 10% SDS, and 0.03% glycerol. The products
were separated on 1.2% agarose gel and visualized by autoradiography.Cleavage Reaction
TFO-OPC conjugates were mixed
with LTR U3 or LTR U5 substrate (10 nM) under the same
conditions used for the integration reaction. The reaction was stopped
by adding 10 mM EDTA and formamide. Cleavage products were
subjected to electrophoresis in 18% denaturing polyacrylamide gel and
visualized by autoradiography. Autoradiographs from the cleavage
reaction experiments were analyzed using a Bio-Profil (Vilber Lourmat)
microdensitometer. For each band corresponding to cleavage products, we
calculated the fraction F = I/I
,
corresponding to the relative inhibition, where I is the
integrated volume of the cleavage product in the presence of increasing
concentration of HIVS-OPC and I the value of the
same band without conjugate. Relative inhibition was plotted versus HIVS-OPC concentration, and data were fitted using a nonlinear
least-squares fitting procedure of INPLOT4 software. Midpoint of
integration efficiency was calculated for F = 0.5,
determined in the best fit.Electron Microscopy
3`-Biotinylated
oligonucleotide bio-3`-GGTTTTTGTGT-5`-NH was purchased from
Eurogentec and conjugated with OPC as described elsewhere(23) .
pU5 HIV plasmid DNA carrying the U5 LTR end was obtained by the
following method. A 42-mer target used for footprinting experiments was
modified by addition terminal nucleotides in order to obtain EcoRI and HindIII cleaved ends and cloned into EcoRI-HindIII sites of the pSP65 vector. For electron
microscopy, a procedure similar to that described in Cherny et al.(27) was used. 0.2 µg of pU5/NheI plasmid DNA
was incubated in a 10-µl volume with bio-oligo at room temperature
for 1 h in a buffer containing 10 mM Tris-acetate, pH 7.2, 20
mM sodium acetate, 5 mM MgAc, 5 mM MnCl, 0.1 mM spermine. The final
concentration of oligonucleotide was 1 µM. After
incubation, the mixture was passed through the Superose 6 column
equilibrated with 10 mM Tris-acetate, pH 7.2, 20 mM sodium acetate, 10 mM MgCl. The
DNA-containing fractions were collected and streptavidin (Sigma) was
added to a final concentration of 5-20 µg/ml (80-300
nM). After a 10-min incubation at room temperature, the gel
filtration step was repeated. A 5-µl aliquot was then applied to a
carbon film glow discharged in the presence of pentylamine vapors
according to Dubochet et al.(28) , stained with
0.5-1% aqueous solution of uranyl acetate, and rotary shadowed
with tantalum/tungsten with an electron gun of a Balzers MED 010
apparatus. The samples were observed with a Zeiss CEM-902 electron
microscope in the annular dark-field mode according to Delain et
al.(29, 30) . Image recording and length
measurements of DNA molecules were performed with the built-in Kontron
image analyzer system and software.
)
M. B. thanks Dr. Serge Fermandjian for manuscript
preparation, Eliane Franque for skillful technical assistance, and
Frédérique Subra and
Pascale Bouillé for helpful discussions. D. I. C.
thanks Dr. E. Delain for his help and encouragement in this work.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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