J. gen. Virol. (1981), 54, 425-429
425
Printed in Great Britain
The Distribution of Guanine-Cytosine Pairs in Adenovirus DNAs
(Accepted 30 January 1981)
SUMMARY
The distribution of guanine-cytosine (GC) pairs in the DNA of the highly
oncogenic simian adenovirus type 7 (SA7) and the non-oncogenic human adenovirus
type 6 (Ad6) has been studied by thermal denaturation and CsC1 density-gradient
centrifugation. The differential of the DNA thermal denaturation curves shows the
presence of pronounced peaks which indicates uneven distribution of GC pairs along
the DNA chains and the presence of regions with GC content from 30 to 74% in
SA7 D N A and from 40 to 68% in Ad6 DNA. The DNA restriction fragments
obtained by treatment with EcoRI, BamHI, SalI, BgllI and HindlII were subjected
to CsC1 density-gradient centrifugation. GC content of the fragments ranged from 45
to 70% for SA7 DNA and from 43 to 61% for Ad6 DNA. The GC content of the
extreme left-hand fragments, where the transforming gene(s) is located, was higher
than the average for SA7 DNA and lower than the average for Ad6 DNA. The most
GC-rich regions were localized in the centre of the genome. The GC content of the
right-hand part of both viral genomes was lower than the average.
Although the complete nucleotide sequence of some small viral genomes has been
determined, this type of analysis still represents a major scientific undertaking needing many
months work by a large research team. Therefore, a less detailed analysis of the DNA
primary structure is still important, for example the determination of GC distribution along
D N A chains.
Such an approach is of special value when it is coupled with the determination of genome
functions or the classification of a virus. For example, the GC content of the terminal
fragments is one of the criteria for the orientation of the molecule of human adenovirus
(Bachenheimer et al., 1977). In addition, considerable attention has been focused on the
correlation between the primary structure of DNA and the oncogenicity of adenoviruses. For
human adenoviruses, oncogenicity is enhanced with the reduction in the content of GC pairs
(Pina & Green, 1965), whereas for simian adenoviruses the reverse is true (Goodheart, 1971).
Since this correlation is based solely on the estimation of the overall nucleotide composition
of the DNA, we decided to carry out a more detailed analysis, particularly to define the
distribution of GC pairs in DNA of human and simian adenoviruses which differ in their
oncogenicity.
SA7 and Ad6 viruses were grown and purified as described previously (Naroditsky et al.,
1978; Dubichev et aL, 1978). Conditions for the isolation of DNA, endonucleases EcoRI,
BamHI, SalI, BgllI and HindIII, the digestion of DNA and the isolation of restriction
fragments were the same as described previously (Naroditsky et al., 1978, 1980; Dimitrov et
al., 1979).
Centrifugation of DNA and its fragments in CsCI density gradient was performed in an
AnG-T rotor on a Spinco E centrifuge with a scanning system at 44 000 rev/min for 24 h at
25 ° C (wavelength 264.5 nm). Clostridium perfringens DNA (density in CsC1 1.6911 g/ml)
and Micrococcus lysodeikticus DNA (density in CsC1 1.731 g/ml) were used as references.
Buoyant densities and GC contents of DNA and its fragments were calculated as described
by Erikson & Szybalski (1964) and De Ley (1970) respectively. Melting of DNA in 0.1 ×
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I
0.2
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Temperature (°C)
Fig. 1. The differential melting curves of SA7 and Ad6 DNAs. The solvent used was 0.1 x SSC.
(a) SA7, (b) Ad6.
standard saline-citrate buffer (SSC) was performed in a modified precision Hitachi ED 20
spectrophotometer as described by Lyubchenko et aL (1976). Differentiation of the melting
curves was accomplished by a graphic method.
For an analysis of the GC content of D N A molecules we used two, approaches: the
estimation of the differential melting curves of D N A and the measurement of buoyant
densities of D N A fragments obtained by treatment with restriction endonucleases. Ad6 D N A
has an average GC content of 53.3% and a mol. wt. of 23.3 x 106 (Dubichev et aL, 1978).
SA7 D N A has an average G C content of 57.6% and a mol. wt. of 22.5 x 106 (A. G.
Dubichev, unpublished data).
The differential melting curves of SA7 and Ad6 DNAs are represented in Fig. 1, which
indicates the uneven distribution of GC pairs among the different parts of the genome. From
the curve pattern it can be seen that melting of the largest part of the D N A molecule occurred
in the temperature range of 70 to 82 °C, which corresponds to the existence of long regions
(blocks) with a G C content of 40 to 65 % (Lyubchenko et al., 1976; Pavlov et al., 1977).
However, relatively small regions of the viral D N A s melted outside this temperature range.
The peak at 85 °C in SA7 D N A indicates the existence of especially refractory regions with a
G C content up to 74 %. Ad6 D N A has a GC-rich fraction, but with a lower content of G C
pairs (68 %) compared with SA7 DNA. From the ratio of the melting peak area to the whole
differential curve area one can calculate that the GC-rich fractions consist of about 1000
nucleotides. The peak (68.4 °C) at the beginning of the melting curve corresponds to regions
of SA7 D N A with a low thermostability which were completely melted out at more than 2 °C
lower than the major part of DNA. The GC content in the early melted regions is 30 + 4%,
determined from the 260/280 nm ratio of the first peak. The ratio of area under the first
melting peak to the whole differential curve area indicates that the early melted region
consists of 350 nucleotide pairs. This estimate is supported by the small width of the
transitions during the melting of this region. One can calculate that a transition width of
0.3 °C corresponds to the cooperative melting of a region of 300 to 400 nucleotide pairs
(Lyubchenko et al., 1976; Pavlov et al., 1977). In contrast to SA7 DNA, Ad6 D N A had no
early melted parts which were melted out before melting of the major part of DNA.
EcoRI, B a m H I and SalI endonucleases cleave SA7 D N A into two, seven and six
fragments respectively (Naroditsky et al., 1978), designated by latin letters in the order of
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427
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I
I
70
i
I
I~
65
60
55
50
45
I
40
60
80
Genome length (%)
G C F D
A
20
B
l
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II
II
C
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FDE
II
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1
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D
II
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,
BamHI]
SalI I
BgllI ~ SA7
EcoRIJ
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100
A
EcoRI
BamHI1
HindlII Ad6
SalI
Fig. 2. Distribution of GC pairs along SA7 ( - - ) and Ad6 (-..) DNA molecules. Dashed lines mark
mean GC content. Restriction maps for SA7 and Ad6 DNAs are given below.
decreasing mol. wt. The buoyant density of all EcoRI, BamHI, SalI fragments and of the D,
E, F fragments obtained from the SalI C fragment after redigestion by R. BgllI (Zavizion et
aL, 1979) was determined by CsCI density-gradient centrifugation. We also determined the
buoyant density of the Ad6 DNA fragments obtained by the combined cleavage by EcoRI
and BamHI endonucleases. Each of the endonucleases cleaved Ad6 DNA at three sites,
producing seven fragments. We also defined the buoyant density of all SalI fragments of Ad6
DNA (Naroditsky et al., 1980) with the exception of SalI D fragment, which was omitted
due to its small size (about 1.5% of the Ad6 genome) and difficulties in its preparative
isolation. The BamHI B fragment, carrying the transforming gene(s) was additionally cut by
endonuclease HindlII into F, C 1 and Ared fragments and their buoyant densities were also
determined.
Since the order of the DNA fragments is known and their GC content has also been
determined, it is easy to build up the distribution of the GC pairs along the DNA chain. As
seen in Fig. 2, SA7 and Ad6 DNAs show a rather asymmetrical distribution of GC pairs.
In general, it should be noted that the right half of the SA7 and Ad6 molecule contains
fewer G C pairs than the left half, where the transforming gene(s) is located (Ponomareva et
al., 1979). Attention should be drawn to two central regions with extremely high GC content,
one of them obviously corresponding to the most refractory peak of the melting curve (Fig.
1). The central steep depression, with a minimum of GC pairs in the case of SA7 DNA, does
not fit into the early melting region in Fig. 1. One of us (A. S. Borovik & Yu. L. Lyubchenko,
unpublished data) while constructing an electron microscopic denaturation map of SA7 DNA
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428
Short communications
was able to localize the early melting peak (TreeI 68.3 °C). This particular region was located
at a 0.25 distance from the right end of SA7 DNA corresponding to the Sail A fragment.
It should be noted that the transforming gene(s) region in both DNAs is characterized by a
slight excess of GC pairs in comparison with the average content.
The average distribution analysis confirmed once again the validity of the division of the
adenovirus genome into left, central and right sections. In general, the left half of the
adenovirus genome was characterized by an enhanced GC content in comparison with the
right half. Thus, the generally accepted criteria for the orientation of a number of human
adenovirus genomes (Bachenheimer et al., 1977) can be widened to include simian and other
human adenoviruses which have not been studied in detail before.
The findings presented here allow a more detailed analysis of the GC pair distribution
along a D N A molecule and of its relationship with the function of the transforming gene(s)
region. In the light of recent findings on the transforming a n d tumourigenic activity of
fragments of adenovirus DNA, one might expect that the primary structure of the
transforming gene itself or its immediate surroundings are responsible for the relationship
between oncogenicity and the GC content. However, as can be seen from our data, it is not
so. The SA7 transforming gene(s) is located in BgllI F + D fragments with a mol. wt. of 1.8
× 106 (Zavizion et al., 1979; T. I. Ponomareva, unpublished data) and has a GC content of
59.5%, which is only 2% higher than the average GC content of the whole DNA. The
Ad6 transforming gene(s) which is located in the HindlII F fragment (Tikchonenko et aL,
1980), has a mol. wt. of 1.7 × 106 and a GC content of 50.4%, which is 3% lower than the
average GC content of Ad6 DNA. The reports of Van Ormondt et al. (1978), Sugisaki et al.
(1980) and Dijkema et al. (1980), who have determined the nucleotide sequence of the left
terminal fragment of adenovirus DNA type 5, type 12 and type 7, leads to the same
conclusion. It can be calculated that the average GC content in the left terminal fragment of
the DNA is lower than the average value for the whole DNA" the Hpa E fragment of Ad5
DNA is 51% GC while the whole Ad5 DNA is 56 %, the BgllI H fragment of Ad7 DNA is
49% GC while the whole Ad7 D N A is 53% and the HindlII G fragment of A d l 2 DNA is
46% GC while the whole Adl2 DNA is 47%. Although these deductions remain to be
supported by additional studies, it appears that the Green-Goodheart rule relates not to
primary structural features of the extreme left region of the adenovirus genome but rather to
its central and right regions. The transforming gene(s) comprises too small a part of the viral
genome for its primary structure to markedlyaffect the average GC content in the DNA.
According to our data the GC content in the left terminal fragments did not differ
significantly from the average value. In contrast, the right and the central regions were
characterized by a more prominent deviation of the GC content from the average, and they
may be more significant for oncogenicity. This poses the following question: how are these
genome regions related to the degree of oncogenicity if they are not needed for the
manifestation of the transforming and tumourigenic activity?
1D.L Ivanovsky Institute of Virology
U.S.S.R. Academy of Medical Sciences
2Institute of Molecular Genetics
U.S.S.R. Academy of Sciences
Moscow, U.S.S.R.
T. I. TIKCHONENKO 1.
A. G. DUBICHEV1
YU. L. LYUBCHENKO 2
N. P. KVITKO 2
N. M. CHAPLYGINA 1
T. I. KALININA 1
R. S. DREIZIN~
B. S. NARODITSKY 1
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429
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