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INTRODUCTION |
Alternative splicing generates different mRNA species from a
common precursor molecule, and these mRNAs may encode proteins that
differ in their biochemical and biological properties. One example for
this phenomenon is vascular endothelial growth factor (VEGF)1 which is an important
regulator of angiogenesis and blood vessel permeability (1, 2). Of its
three major splice forms only VEGF165 and
VEGF189 bind heparin, whereas VEGF121 does not.
Moreover, VEGF165 and VEGF121, but not
VEGF189, are secreted from cells. Two further splice
variants, VEGF145 and VEGF206, have been found at low abundancy in some tissues (3, 4). All VEGF isoforms contain the
amino acids encoded by the exons 1-5 and by exon 8 of the VEGF gene.
Exon 6 is present in VEGF145, VEGF189, and
VEGF206. Only VEGF189 and VEGF206
contain exon 7. An extension of exon 6 because of utilization of an
alternative splice donor site is specific for VEGF206.
All variants of alternatively spliced mRNAs can be detected
simultaneously by reverse transcription-polymerase chain reaction (RT-PCR) analysis if the annealing sites of the two primers are selected at both sides of the facultative exons in exons common to all
transcripts. The resulting DNA products corresponding to the different
mRNA variants are usually identified by size and/or by Southern
hybridization. We found that RT-PCR analysis of alternatively spliced
genes is complicated by the appearance of previously unrecognized DNA
complexes which are composed of amplification products from different
transcripts. We have characterized the structure of heteroduplexes with large single-stranded loops and have
shown that these heteroduplexes form novel four-stranded
DNA-complexes.
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MATERIALS AND METHODS |
RT-PCR--
Total RNA was extracted from the human epidermoid
cell line A431 with RNazol (Cinna/MRC, Cincinatti, Ohio) and
reverse-transcribed as described previously (2). For amplification of
VEGF-specific cDNAs, a sense primer annealing within exon 1 (5'-CCA
TGA ACT TTC TGC TGT CTT-3') and an antisense primer derived from the
untranslated region of exon 8 (5'-TCG ATC GTT CTG TAT CAG TCT-3') (2)
were used in a reaction involving denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for
1 min. The number of cycles was varied from 25 to 40. PCR products were
electrophoresed on a 1.5% agarose gel containing ethidium bromide.
DNA Preparation from Agarose Gels--
DNA was prepared from
bands of tris borate-EDTA-agarose gels with the Qiaex II gel extraction
kit (Qiagen, Hilden, Germany) according to the instructions of the
manufacturer. DNA was eluted from Qiaex particles with water at a
temperature of 50 °C for 15 min.
S1 Analysis--
50 ng of DNA were incubated with 20 units of S1
nuclease (Boehringer Mannheim, Germany) in S1 buffer (33 mM
Na-acetate, 50 mM NaCl, 0.03 mM
Zn2SO47H2O, pH 4.5) for 1 h at
37 °C. Reaction products were separated electrophoretically on a 2%
agarose gel and blotted onto a nylon membrane. For Southern
hybridization, the oligonucleotides used as PCR primers were
32P-labeled with terminal transferase (Stratagene,
Heidelberg, Germany) and incubated with the blot at 65 °C for 2 h. Membranes were washed and exposed to x-ray film (Kodak, Rochester, NY).
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RESULTS |
Detection of Aberrant RT-PCR Products--
To detect VEGF splice
variants expressed at low levels, PCR was performed on cDNA from
A431 cells at high sensitivity conditions. In addition to the
previously described isoforms VEGF121, VEGF165, and VEGF189, (2) which yield amplification products of 526, 658, and 730 bp, respectively (Fig.
1A), an additional band
appeared when the number of PCR cycles was raised over 30 (Fig.
1B). This DNA band had an apparent size of approximately 600 bp on a 1.5% agarose gel. Compared with the other bands, its abundancy
increased overproportionally during the last PCR cycles (Fig.
1B). These kinetics suggested that the mechanism of its
formation was different from the one that generates standard PCR
products. Southern hybridization with an oligonucleotide recognizing
all known splice forms of VEGF confirmed that the 600-bp band
originated from VEGF cDNA (data not shown). An additional band of
the apparent size of 1200 bp was found when we tried to maximize the
amplification yield by combinations of high cycle number, high amounts
of cDNA template, and high polymerase concentrations (see
below).

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Fig. 1.
RT-PCR amplification of VEGF mRNA splice
variants. A, structural organization of the three major
VEGF splice forms expressed by A431 cells. Exons are shown as
boxes and are not drawn to scale. The position of the PCR
primers is indicated by arrows. B, agarose gel
electrophoresis of VEGF PCR products derived from A431 cDNA. VEGF
RT-PCR was performed as described under "Materials and Methods."
Aliquots of a common reaction mixture were distributed to a series of
tubes that were removed from the thermocycler after 25, 28, 31, 34, 37, and 40 PCR cycles (lanes 1 to 6, respectively).
Equal volumes were loaded on a 1.5% agarose gel containing ethidium
bromide. The position of the aberrant PCR product is indicated by an
arrow. Sizes of a DNA length marker (M) are shown
in base pairs.
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Identification of the Components of the Aberrant RT-PCR
Product--
For further analysis, the 600-bp band was purified from
an agarose gel as described under "Materials and Methods." When its DNA strands were melted at 95 °C and allowed to reanneal, three DNA
species were observed: the 600-bp band and the bands corresponding to
the size expected for the VEGF121 and VEGF165
amplification products (Fig.
2A). The identity of the two
latter DNA species was confirmed by sequencing. To test whether these
two components were sufficient for the formation of "600-bp DNA",
purified VEGF121 and VEGF165 PCR products were
mixed, denatured, and renatured. An additional band with the apparent
size of 600 bp was obtained (Fig. 2B). These data
demonstrated that the 600-bp band represents a heteroduplex of
VEGF121 and VEGF165 strands.

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Fig. 2.
Denaturation and renaturation of RT-PCR
products. A, DNA prepared from the PCR gel band with an
apparent size of 600 bp (lane 1) was denatured by heating to
95 °C for 5 min and then cooled down slowly to room temperature
(lane 2). Subsequently, products were electrophoresed as in
Fig. 1B. B, DNA was prepared from the bands corresponding to
VEGF165 (lane 1) and VEGF121
(lane 2). DNAs were treated as in panel
A, either separately (lanes 1 and 2)
or after combining in one volume (lane 3). Lanes
M are DNA size markers.
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S1 Nuclease Analysis of the VEGF121/VEGF165
DNA Heteroduplex--
To elucidate the structure of the
VEGF121/VEGF165 heteroduplex, DNA from the
600-bp band was digested with S1 nuclease. The cleavage products were
separated by agarose gel electrophoresis, blotted onto a nylon
membrane, and hybridized with radioactively labeled oligonucleotides.
To specifically recognize cleavage products containing the 5' or the 3'
terminus of the VEGF gene, the corresponding PCR primers were used as
probes. Using the PCR primer annealing at the 5' end of VEGF, we
obtained a signal corresponding to a fragment of 420 bp in length that
was not detectable with the 3' probe (Fig.
3, A and B,
lanes 2). This size equals the length of the sequence common
to all splice variants at the 5' end of the gene (Fig. 3C).
By hybridization with the 3' primer, a 100-bp band corresponding to the
stretch of sequence identity at the 3' end of VEGF121 and
VEGF165 was detected (Fig. 3B). From the localization of S1 cleavage site at the position of the
VEGF165-specific exon 7, it became evident that this region
is single-stranded in the complex. Both the 100- and the 420-bp band
are generated by cleavage of both heteroduplex DNA strands at the site
of sequence divergence. Because of incomplete digestion, an
intermediate product, in which only the exposed single-stranded loop
but not the second strand of the complex is cleaved, was present in the
resulting reaction mixture. Both probes hybridized to that intermediate of 520 bp length (Fig. 3, A and B). These results
suggest that the VEGF165-derived heteroduplex DNA strand
adopts an
-like conformation in which the sequence corresponding to
exon 7 forms a single-stranded loop (Fig. 3C).

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Fig. 3.
S1 analysis of heteroduplex DNA.
Heteroduplex DNA composed of VEGF165 and
VEGF121 PCR products (lanes 2) was digested with
S1 nuclease and electrophoresed in parallel with an undigested aliquot
(lanes 1) and VEGF121 PCR product (lanes
3). Blots were hybridized either with radioactively labeled 5' PCR
primer oligonucleotide (panel A) or with the 3' PCR primer
oligonucleotide (panel B). The sizes of the bands as
determined by comparison with a DNA length standard are shown on the
left side. Heteroduplex-derived bands specifically
hybridizing with one of the two oligonucleotides are indicated by
arrows. C, schematic representation of the VEGF
PCR products involved in heteroduplex formation and of the two types of
heteroduplexes. DNA strands are represented by lines with
arrowheads at their 3' end to indicate their orientation.
The segment of VEGF165 corresponding to exon 7 is shown as
a bold line. Interactions between heteroduplexes that
mediate the formation of a heteroduplex-duplex are indicated by
double-headed arrows.
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Because either the sense or antisense strand of both
VEGF121 and VEGF165 PCR products can
participate in heteroduplex formation, two types of complexes appear.
In one of them, the VEGF165-derived exon 7 sequence is
exposed in sense orientation. In the other heteroduplex-type, the
single-stranded loop is in antisense orientation (Fig. 3C).
The sequence complementarity in the loops suggests that two
heteroduplexes might interact through base pairing in that region (as
depicted by double arrows in Fig. 3C).
Identification of the Second RT-PCR By-product as a Complex of
Heteroduplexes--
The second aberrant PCR product (1200-bp band),
which was detected after maximized DNA amplification (Fig.
4A), was assumed as a
potential complex of two VEGF121/VEGF165
heteroduplexes because it was exactly twice as large. To investigate
whether this product represented a complex stabilized by weak
interactions such as local base pairing, PCR products were subjected to
mild heat denaturation (heating to 70 °C for 10 min and subsequent
chilling on ice). Whereas perfectly double-stranded DNA species and the
VEGF121/VEGF165 heteroduplex remained
unaffected, the 1200-bp band disappeared (Fig. 4A,
lane 2). When, after purification, DNA from this band was
analyzed by gel electrophoresis, only a 600-bp band was found repeatedly (data not shown). In contrast, when DNA derived from the
600-bp band was incubated at high concentrations prior to electrophoresis, a 1200-bp band appeared. Again, heating to 70 °C
and rapid chilling abolished that DNA form and led to a proportional increase in the amount of heteroduplex DNA (Fig. 4B). These
findings strongly suggest that this DNA species represents a novel
complex of DNA heteroduplexes.

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Fig. 4.
Interconvertibility of RT-PCR
by-products. A, VEGF RT-PCR products were either kept
at 4 °C (lane 1) or heated to 70 °C for 10 min
followed by chilling on ice (lane 2). B, DNA
prepared from the PCR band with the apparent size of 600 bp was either
kept at 4 °C (lane 1) or heated to 70 °C for 10 min
followed by chilling on ice (lane 2). Lanes
M are DNA size markers.
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DISCUSSION |
The generation of DNA heteroduplexes in experimental procedures
has been described previously. Methods for the detection of genomic
mutations such as AMD (amplification and mismatch detection) are based
on the annealing of sample-derived PCR products with nonmutated bona
fide DNA (5). In these analyses, the heteroduplexes consist of DNA
strands that differ only by single bases. The formation of
heteroduplexes with long unpaired segments has been observed as a
complication of quantitative PCRs that use an artificial internal
standard with partial sequence identity to the target DNA (6-8). Here
we show that, in the analysis of genes with alternatively spliced
transcripts, heteroduplex formation is a problem even where only
qualitative aspects are concerned. We detected heteroduplexes after
RT-PCRs of several alternatively spliced mRNAs (data not shown)
such as those of the apoptosis regulator bcl-x (9) and of caspase 10 (10). Of particular importance is the appearance of the
VEGF121/VEGF165 heteroduplex because, under
standard agarose gel electrophoresis conditions, this complex has the
same migration rate as the amplification product of a recently
described minor splice variant, VEGF145 (3). To investigate
the significance of VEGF145 expression, we avoided the
formation of the VEGF121 and VEGF165
amplification products by selecting a 5' primer that annealed within a
part of exon 6 that is present in VEGF189 and VEGF145 but absent from VEGF121 and
VEGF165. When cDNA from the A431 cells was used, the
band corresponding to VEGF145 was much less abundant than
the VEGF189 band. This observation contradicts results
presented by Poltorak et al. (11) who employed an RT-PCR that allows for the formation of the
VEGF121/VEGF165 heteroduplex. In light of our
findings, it seems likely that the band obtained by Poltorak et
al. (11) did not originate from VEGF145 but rather represents a VEGF121/VEGF165 heteroduplex.
Similar to the amplification products of VEGF121 and
VEGF165, VEGF189 must be expected to
participate in heteroduplex formation. We could detect a heteroduplex
of VEGF145- and VEGF189-derived DNA strands in
the PCR reaction specific for those two splice forms but not when
VEGF121 and VEGF165 amplification products were
present (not shown). This was probably because of co-migration of small
amounts of heteroduplexes containing VEGF189 with other PCR
products in agarose gel electrophoresis.
In addition to the DNA heteroduplex, a second DNA species was
identified as a by-product of RT-PCR of alternatively spliced transcripts and was found to be a complex composed of two
heteroduplexes. To our knowledge, this is the first report in which
such a DNA heteroduplex-duplex is described. Based on the structural
information obtained by S1 analysis of the heteroduplex, we conclude
that this complex represents a novel DNA structure that is formed by base pairing of sequences exposed in single-stranded loops. Because of
the limited length of the accessible complementary sequences, it is
less resistant to denaturing conditions such as heating to 70 °C,
but was nevertheless found to be stable at 4 °C as well as at room
temperature. Because DNA heteroduplex structures are formed during
recombination processes and bulging out of noncomplementary segments
has been proposed by current recombination models (12), it is tempting
to speculate that interactions between DNA single-stranded loops could
occur in vivo. Similar interactions between short single-stranded RNA loops have been implicated in the regulation of the
replication of the bacterial plasmid ColE1 and in the initiation of
dimerization of retroviral genomes (13, 14). Further studies are
necessary to evaluate the importance of single-stranded DNA loops both
in physiological and in experimental settings.