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Analysis of DNA from human adenovirus type 6 with restriction endonucleases HindIII, BglIII and BamHI
214
Biochimica et Biophysica Acta, 606 (1980) 214--227
© Elsevier/North-Holland Biomedical Press
BBA 99589 ANALYSIS OF DNA FROM HUMAN ADENOVIRUS TYPE 6 WITH RESTRICTION ENDONUCLEASES HindIII, BglIII and BamHI
B.S. NARODITSKY a, T.I. KALININA a, E.Z. GOLDBERG a, A.S. BOROVIK b , E.V. KARAMOV a and T.I. TIKCHONENKO a,. a D.I. Ivanovsky Institute o f Virology, the USSR Academy o f Medical Sciences, Gamaleya str. 16, Moscow 123098 and b Molecular Genetics Institute, the USSR Academy o f Sciences, Moscow (U.S.S.R.)
(Received January 15th, 1979) (Revised manuscript received July 4th, 1979) Key words: DNA map; Restriction endonuclease; (Adenovirus)
Summary The genome of the type 6 h u m a n adenovirus has three restriction sites for R.BamHI, thirteen for R.HindIII and ten for R.BglII. The terminal fragments of DNA cleaved by each of the enzymes have been determined by means of terminal nucleotidyl transferase and by analysis of the DNA-terminal protein complex. The sequence of the cleaved fragments has been determined by partial cleavage of DNA, simultaneous digestion of DNA with various combinations of enzymes and secondary digestion of individual isolated fragments with other enzymes. The following order of the cleaved fragments in the adenovirus t y p e 6 genome has been found (the figures in brackets are the weights in megadaltons): for R.BamHI-B(7,1)-D(3.0)-C(4.05)-A(8.5); for R.HindIII-F(1.7)Cl (2.14)-A(3.44)-M(0.046)-I(1.24)-J(0.77)-D(2.1)-E(1.96)-B(3.18)-H(1.36)L(0.18)-C2(2.14)-G(1.44)-K(0.16); for R.BglII-E(2.07)-B(3.5S)-A(4.8)-C(3.36)I(0.7S)-D(3.25)-G(1.37)-J(0.21)-F(1.85)-K(0.17)-H(0.94).
Introduction The h u m a n adenovirus of the type 6 belongs to the group of nononcogenic h u m a n adenoviruses [2] but it can induce in vitro transformation of some * TO w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . A b b r e v i a t i o n s : R . E c o R I , R . B a m H I , R . H i n d l I I a n d R . B g l I I - r e s t r i c t i o n e n d o n u e l e a s e s f r o m Escherichia coli R Y 13, Baeilus amyloliquefaeiens, Haemophilus inl'luenzae R d a n d Bacillus globigii, r e s p e c t i v e l y . T h e r e s t r i c t i o n e n d o n u c l e a s e s are c l a s s i f i e d a c c o r d i n g t o t h e n o m e n c l a t u r e [ 1 ] . 3 2 p - d N T P , d e o x y t r i p h o s phage labelled w i t h r a d i o a c t i v e p h o s p h o r u s at t h e a p o s i t i o n .
215
cells [3]. The adenovirus type 6 genome is a linear double helical DNA molecule with the molecular weight about 23 • 106 (see below). G + C content in adenovirus type 6 DNA is 52.6% (Tikchonenko, T.I., unpublished results) which is lower than the value {56--60%) that should be revealed in adeno DNA in accordance with Green rule [4]. In contrast to other adenoviruses [5,6] of this group, t o o few studies have been made concerning the physicochemical properties of the adenovirus type 6 DNA and its cleavage with restriction endonucleases. We have reported earlier [7,8] on the number of recognition sites and the sequence of cleaved fragments in adenovirus type 6 DNA for R.EcoRI and R.SalI. Later our results for the R.EcoRI fragments have been confirmed b y Forsblom [9], who used another technique for mapping the DNA fragments. This paper presents the results of mapping of the adenovirus t y p e 6 DNA fragments cleaved with three other restriction enzymes, namely R.BamHI, R.HindIII and R.BglII. Materials and Methods
Cell culture, virus growth and DNA extraction. The adenovirus type 6 was propagated in HeLa cells of the Bristol line [10]. The virus was extracted from the infected cells by freezing and thawing out. The extracted virus was treated with Freon-113 and concentrated by centrifugation on the CsC1 cushion (p = 1.43 g/cm 3) according to the procedure [11]. The concentrated viral suspension was further purified by t w o cycles of equilibrium centrifugation in CsC1 density gradient [12]. Electron microscopy was used to control the purity of the viral preparation. The viral DNA was isolated according to the modified p r o c e d u r e [13]. Source o f enzymes. The endonuclease R.EcoRI was isolated from the E. coli RY-13. The restriction enzyme was purified according to the modified procedure [14]. The enzyme R.BamHI was isolated according to the procedure [15]. The enzyme R.HindIII was supplied b y Miles (U.S.A.) and R.BglII was isolated according to the procedure [16]. The media for the enzyme reactions were: for R.EcoRI: 70 mM Tris-HC1, pH 7.5/50 mM NaC1/10 mM MgC12/7 mM 2-mercaptoethanol [14]; for R.BamHI: 6 mM Tris-HC1, pH 7.4/6 mM MgC12/ 6 mM 2-mercaptoethanol [17]; and for R.HindIII and R.BglII: 10 mM TrisHC1, pH 7.5/6.0 mM MgC12/10 mM KC1/6 mM 2-mercaptoethanol [6]. The temperature was 37°C, the time of incubation was 15--90 min, the volume of the reaction mixture was 20--50 pl, the DNA content was 0.5--2 pg and the a m o u n t of enzyme varied from 0.5 to 5 ~1. To produce partial cleavage the viral DNA was incubated either with diluted enzyme or in a medium with a higher ionic strength at 30°C. The enzyme reaction was stoped b y adding EDTA or incubating the mixture for 5 min at 65°C. Electrophoretic analysis. The cleaved fragments were analyzed by agarose gel electrophoresis [18,19] in tubes or in 3--5 mm slabs of agarose gel (Bio-Rad, U.S.A.). The gel was stained in 1/~g/ml solution of ethidium bromide (Calbiochem, U.S.A.) for 20 min and ultraviolet photographs were used to detect the positions of the DNA fragments. The internal standard for calculating the molecular weights of the cleaved fragments was the R.EcoRI fragments of the phage ~ DNA, the molecular weights of which were taken from Ref. 20. In electrophoresis of labelled material the gels after staining with ethidium
216 bromide were either used for exposing high-sensitivity X-ray film or cut into 1-mm disks which were put into vials for measuring radioactivity, dissolved with the Soluen universal solvent (0.4 ml per disk) and stored for 2 h at 60°C. Then 10 ml of toluene scintillator were added and the activity was measured with an SL-30 scintillation counter (Intertechnik, France). Analysis of the adenovirus type 6 DNA fragments in the agarose-polyacrylamide copolymer was conducted according to the procedure [21]. Electron microscopy. The DNA preparations were made according to the modified formamide procedure [22]. 25 ~l of c y t o c h r o m e c, cleaved with 10 t~g/ml BrCn [23], and 25 ~1 of 10 pg/ml DNA were added to 50 t~l of the buffer solution containing 0.01 M Na3EDTA, 0 . 0 4 M phosphate buffer (pH 7.0) and 90% formamide (Merck, F.R.G.). Deionized water was used as the hypophase. The protein film on the water surface was applied to a copper grid coated with a thin carbon film. The specimen was shadowed with platinum and photographed in a JEM-7 electron microscope. The magnification was calibrated by the replica of the diffraction grating. Elution o f DNA from the agarose gel. Following electrophoresis of DNA the agarose gel was stained with ethidium bromide and the gel band containing a given DNA fragment was cut off, frozen, placed into a centrifuge test-tube and centrifuged in a SW-50.1 rotor for 1 h at 30°C and 35 000 rev./min in an L-5-50 centrifuge (Beckman, U.S.A.). The supernatant was collected and deproteinized 2 or 3 times, and the DNA was precipitated with ethanol [24]. Preparation o f the 32p-labelled adenovirus type 6 DNA. The 32p-labelled DNA was obtained either in vivo according to the procedure [25] with the specific activity of 2 • 10 s cpm/~g or by the nick-translation reaction [26]. In the latter case the specific activity of the preparation was 1 • 106--1.2 • 106 cpm/~g. The 3:P-labelled dATP (Amersham, U.K.) was used as the labelled precursor. Preparation o f the adenovirus type 6 DNA-terminal protein complex. The DNA-protein complex was prepared from purified viral suspension in the presence of 4 M guanidine chloride and the complex was purified by CsC1 equilibrium centrifugation in the presence of guanidine chloride [27 ]. Results and Discussion
Cleavage and mapping o f the R.BamHI fragments o f the adenovirus type 6 DNA Complete cleavage of the adenovirus type 6 DNA by R.BamHI gives rise to four fragments, A, B, C and D (Fig. 1.5). Table I presents the molecular weights of the R.BamHI fragments. The direct electron microscopy determinations of the amounts and lengths of the R.BamHI fragments also reveal the presence of four types of fragment. The histogram in Fig. 2 shows that all the fragments are present in equimolar amounts. The molecular weights of the fragments calculated from the length measurements are in a good agreement with the electrophoretic results; the total molecular weight of the R.BamHI fragments of the adenovirus type 6 DNA (22.9 • 106) agrees with the electron microscopy result (23.1 • 106) for the native adenovirus type 6 DNA and with the total molecular weights of the R.EcoRI fragments (22.76" 106) and the R.SalI fragments (22.85 • 106) of the adenovirus type 6 DNA [7,8].
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Fig. 1. Cleavage of t h e a d e n o v i r u s t y p e 6 D N A b y R . B a m H I a n d a m i x t u r e of R . B a m H I a n d R . E e o R I . Capital l e t t e r s d e n o t e t h e p r o d u c t s of c o m p l e t e cleavage. R o m a n n u m e r a l s d e n o t e t h e p r o d u c t s of p a r t i a l cleavage. T h e D N A f r a g m e n t s w e r e a n a l y z e d in 0.7% agarose slab gel at 1.5 V / c m for 1 8 - - 2 2 h, or in 1% agarose t u b e s . 1. D N A o p r o t e i n c o m p l e x t r e a t e d w i t h R . B a m H I a n d t h e n i n c u b a t e d w i t h 10/~g of p r o n a s e at 3 7 ° C f o r 15 rain. 2. D N A - p r o t e i n c o m p l e x t r e a t e d w i t h R . B a m H I . 3. A d e n o v i r u s t y p e 6 D N A d i g e s t e d u n d e r t h e p a r t i a l cleavage c o n d i t i o n s (0.5/~1 R . B a m H I , 10 rain, 3 2 ° C , 0.3 M NaC1). 4. P r o d u c t s o f s e c o n d azy R . B a m H I cleavage o f the D N A f r a g m e n t s isolated f r o m the b a n d w i t h the m o l e c u l a r w e i g h t 7 • 106. 5. A d e n o v i r u s t y p e 6 D N A digested u n d e r the c o n d i t i o n s of c o m p l e t e cleavage (1 /~1 R . B a m H I , 15 rain, 3 7 ° C ) . 6. T h e R . E c o R I f r a g m e n t s o f the a d e n o v i r u s t y p e 6 D N A . 7. A d e n o v i r u s t y p e 6 D N A c l e a v e d s i m u l t a n e o u s l y b y R . E c o R I a n d R . B a m H I . T h e cleavage c o n d i t i o n s axe t h e s a m e as f o r cleavage b y R . E c o R I . 8. T h e R . B a m H I f r a g m e n t s o f a d e n o v i r u s t y p e 6 D N A .
The order of the R.BamHI fragments of the adenovirus type 6 DNA was determined from the results of partial cleavage of DNA and by identification of the terminal fragments using the analysis of the DNA-terminal protein complex. The terminal fragment covalently linked protein produced in cleavage of the complex does not enter agarose gel [28]. The fragments A and B were not found in the agarose gel after electrophoretic separation of the restriction mixture, while the fragments C and D had the typical mobility in the gel (Fig.
I
a b c d
D E F G H I J K L M
A B C
8.4 7.8 6.4 6.15 5.16 3.35 2.41 0.73 --
29.6 d
16.1 13.9
Distribution of 32p (%)
R.HindIII
MOLECULAR
_+ 0 . 1 5 _+ 0 . 1 2 ± 0.14 X 2
2.1 _+ 0 . 0 9 1.96 ± 0.08 1 . 7 _+ 0 . 1 1 c 1.44 ± 0.06 1 . 3 6 +_ 0 . 0 7 1.24 + 0.09 0 . 7 7 _+ 0 . 0 5 0 . 6 _+ 0 . 0 6 c 0.18 ± 0.01 0.046 ± 0.004 22.4
3.44 3.18 2.14
OF
THE
15.36 14.20 9.55 X 2 (C 1 + C 2 ) 9.38 8.75 7.59 6.43 6.07 5.54 3.44 2.68 0.80 0.21 Z 100%
Percentage of the genome
WEIGHTS
Molecular weight a (X 1 0 - 5 )
fragments
AND
OF
9.72 7.9 6.17 4.51 3.02 0.89 0.66
30.35 d
20.33 16.4
Distribution of 32p (%)
THE
_+ 0 . 0 7 + 0.08 +- 0 . 1 ± 0.12 _+ 0 . 1 1 ± 0.09 _+ 0 . 0 0 5 _+ 0 . 0 0 5
Z 22.37
3.25 2.07 1.85 1.37 0.94 0.78 0.21 0.17
4.8 + 0.2 3.58 + 0.12 3.36 + 0.06
TYPE
~ 100%
14.53 9.26 c 8.72 6.13 4.2 c 3.49 0.98 0.76
21.47 16.01 15.03
Percentage of the genome
ADENOVIRUS
Molecular weight a (X 1 0 - 5 )
R.BglII fragments
FRAGMENTS
2~ 2 2 . 9
2.95
8.87 7.05 4.03
Electron microscopic mol.wt, b (X 1 0 - 6 )
_+ 0 . 1 2
C and D.
~ 22.65
3.0
8.5 ÷ 0.25 7 . 1 _+ 0 . 2 1 4 . 0 5 +_ 0 . 1 5
Molecular weight (X 1 0 -6 )
BY R.HindIII,
fragments
CLEAVED
R.BamHI
6 DNA
The values are means from 7--11 experiments. Measttrements of electrophoretic mobility in agarose and agarose-acrylamide gel. Col EI DNA was used as the internal standard. The end fragments were determined from analysis of the DNA-protein complex. S i n g l e r a d i o a c t i v i t y p e a k s w e r e d e t e c t e d f o r t h e b a n d s c o r r e s p o n d i n g t o t h e R . H i n d I I I f r a g m e n t s C 1 , C2 a n d D a n d t h e R . B g l I l f r a g m e n t s
Fragments
THE NUMBERS R.BamHI
TABLE
13.25
37.53c 31.35 c 17.88
Percentage of the genome
R.BglII AND
b~
219 D
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E 20
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3 Z
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Fig. 2. H i s t o g r a m o f l e n g t h m e a s u r e m e n t s (/~m) f o r t h e R . B a m H I f r a g m e n t s o f a d e n o v i r u s t y p e 6 D N A . P e r c e n t a g e s o f t h e f r a g m e n t s in r e l a t i o n t o t h e t o t a l n u m b e r o f m e a s u r e d m o l e c u l e s : A , 1 9 ; B, 2 2 ; C, 23; a n d D, 2 5 .
1.2). When before electrophoretic analysis the same mixture of fragments was treated with pronase, all four R.BamHI fragments were found in the gel (Fig. 1.1). The 3'-labelling of intact DNA with the use of terminal nucleotidyltransferase was chosen as an independent method for the determination of the end fragments. The fragmentation of the DNA with BamHI and the subsequent fractionation of the hydrolyzate in agarose gel have shown that A and B are the end fragments. The determine the order of the internal R.BamHI fragments in the adenovirus type 6 DNA the optimum conditions were selected for partial cleavage of the DNA. The products of partial cleavage contained, apart from the four fragments (A, B, C and D) produced by complete cleavage, additional fragments denoted as I, II, III, IV and V in the order of decreasing molecular weight (Figs. 1.3, 1.4 and Table II). The molecular weights of these fragments correspond to the sums of weights of the fragments of complete cleavage: AC, ACD, BCD, BD and CD (Table II). To detect the fragment V which has the same molecular weight as fragment B, DNA was eluted from the gel band where the nucleic acid with molecular weight 7" 10 6 was located. After secondary cleavage of this DNA by R.BamHI and separation of the cleaved fragments in the agarose gel, the fragments C and D were found apart from the fragment B (Fig. 1.4). The good agreement between the predicted values and the measured molecular weights of the partial cleavage products (Table II) makes it possible to determine the fragment composition of the partial cleavage products and shows that the fragment D is between the fragments C and B and the fragment C is associated with the fragment A. Thus, the above results on the
220 TABLE
II
MOLECULAR WEIGHTS OF THE TIAL CLEAVAGE WITH R.BamHI
ADENOVIRUS
TYPE
6 DNA
FRAGMENTS
PRODUCED
BY PAR-
The weights are means of the results of six individual experiments. Fragments
Expected tool. weight ( × 1 0 -6 )
I II Ill IV
V
Measured tool. weight ( X I O -5 )
Fragment composition
Overlapping order
15.55
15.8 + 0.3
A,C,D
ACD
14.15
13.9 ÷ 0.2
B,C,D
CDB
12.55 10.10
12.8 + 0.2 9.9 • 0 . I
A,C B,D
AC DB
7.05
7.0 + 0.1
C,D
CD
products of cleavage of the adenovirus type 6 DNA by R.BamHI show clearly the following order of the cleaved fragments in the DNA: A-C-D-B (Fig. 5).
The mutual arrangement of the R.EcoRI and R.BamHI fragments To determine the mutual arrangement of the R.EcoRI and R.BamHI fragments, the adenovirus type 6 DNA was simultaneously treated with R.EcoRI and R.BamHI. The products of the reaction did not contain the R.EcoRI fragment A and the R.BamHI fragment A; the products B, C and D of both enzymes were retained and an additional fragment with the molecular weight 2.6 • 106 was found (Fig. 1.7). The results show that the R.EcoRI fragments B, C and D comprise the part of the adenovirus type 6 DNA which corresponds to the R.BamHI fragment A and the R.BamHI fragments B, C and D are located in the part of the adenovirus type 6 DNA which corresponds to the R.EcoRI fragment A. These results suggest that the restriction sites for R.EcoRI and R.BamHI are at the opposite ends of the DNA molecule (Fig. 5). Cleavage o f the adenovirus type 6 DNA by R.HindIII and the order o f the R.HindlII fragments The enzyme R.HindIII cleaves the adenovirus type 6 DNA into 14 fragments (Table I, Fig. 3). The number of fragments was determined by analysis of 3:p-labelled DNA labelled in vivo or in vitro (nick translation). The fragments from A to K were found in the agarose gel (Fig. 3.2) and the fragment L in the agarose-acrylamide copolymer (Fig. 3.3). For detection of fragment M it was necessary to use highly labelled 32p_ labelled DNA preparation and to conduct electrophoretic separation of restriction fragments in 5% polyacrylamide gel with subsequent autoradiography (Fig. 3.4). Quantitative analysis of the distribution of 32p in the gel after separation of the R.HindIII fragments demonstrated that the third largest band contains two DNA fragments with the same molecular weight (Table I) which we denoted as C~ and C2. The fragment D has a molecular weight which is very close to that of the fragments C, and C2 and it can be separated from them only in the 1.4% agarose gel.
221
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Fig. 3. E l e c t r o p h o r e s i s o f t h e R . H i n d I I I f r a g m e n t s o f t h e a d e n o v i r u s t y p e 6 D N A . Capital l e t t e r s d e n o t e the f r a g m e n t s o f c o m p l e t e cleavage. R o m a n n u m e r a l s d e n o t e t h e p r o d u c t s o f p a r t i a l cleavage. T h e D N A f r a g m e n t s w e r e s e p a r a t e d in 1% or 1.4% a g a r o s e slab gel at 1.5 V / c m f o r 2 0 - - 2 2 h a t 4°C (1, 2, 5), in a g a r o s e - a c r y l a m i d e c o p o l y m e r ( 0 . 5 % agarose + 2.5% a c r y l a m i d e ) at 4 V / c m f o r 2 3 - - 2 4 h [ 3 ] a n d in 5% p o l y a c r y l a m i d e slab gel [ 4 ] . 1. D N A - p r o t e i n c o m p l e x t r e a t e d b y R . H i n d I I I . 2, 5. A d e n o v i r u s t y p e 6 D N A ( 0 . 7 - - 1 ~zg) t r e a t e d b y R . H i n d I I I ( 1 - - 2 /~1) at 3 7 ° C f o r 1 h. 3. A d e n o v i r u s t y p e 6 D N A (5/~g) t r e a t e d b y R . H i n d I I I (4 ~ul) a t 3 7 ° C f o r 2 h. 4. T h e R . B a m H I f r a g m e n t D labelled w i t h 3 2 p in v i t r o ( n i c k t r a n s l a t i o n ) a n d t r e a t e d b y R . H i n d I I I ( a u t o r a d i o g r a p h y ) . 6. S i m u l t a n e o u s cleavage o f t h e a d c n o v i r u s t y p e 6 D N A b y R . H i n d l I I a n d R . B a m H I u n d e r the s t a n d a r d c o n d i t i o n s of R . H i n d I I I cleavage. 7. S i m u l t a n e o u s cleavage of the adenovirus type 6 DNA by R.HindllI and R.EcoRI under the standard conditions of R.HindIII cleavage. 8. T h e R . B a m H I f r a g m e n t D (0.4 #g) t r e a t e d b y R . H i n d I I I u n d e r t h e c o n d i t i o n s o f p a r t i a l cleavage (1 ~1, 3 7 ° C , 1 h).
222
Analysis of the R.HindIII fragments produced by cleavage of the DNAprotein complex in the agarose gel demonstrated the absence of the fragments F and K (Fig. 3.1, Table I) suggesting that they are the end fragments (see above). Various techniques were used for determination of the order of the R.HindIII fragments in the adenovirus type 6 DNA. Firstly, the adenovirus t y p e 6 DNA was simultaneously treated with R.HindIII and R.EcoRI, and with R.HindIII and R.BamHI. When the adenovirus type 6 DNA was treated with R.HindIII and R.EcoRI (Fig. 3.7) all the R.EcoRI fragments and B, one of the C fragments and H R.HindIII fragments disappeared in the mixture. Treatment of the adenovirus type 6 DNA with R.HindIII and R.BamHI led to disappearance of all the R.BamHI fragments and the A, B and D R.HindIII fragments (Fig. 3.5). Since we have mapped the R.EcoRI [7] and R.BamHI fragments (Fig. 5) it may be suggested that the R.HindIII fragments B, H and C are in the right half of the genome and the fragments A and D are in the left half of the molecule. Here we refer to the region of the adenovirus type 6 DNA containing the R.EcoRI fragment A as the left part of the molecule. Since the number of the R.HindIII fragments is relatively large we analyzed the individual adenovirus type 6 DNA fragments produced by R.EcoRI and R.BamHI for which the order of the fragments is known. Cleavage of the R.EcoRI fragment B with R.HindIII gave rise to the fragments G and K and a fragment with the molecular weight 1.4 • 10 6 (Table II). Since the fragment K is the end fragment, the fragment G is adjacent to it. Since the products of cleavage of the R.BamHI fragment A with R.HindIII contained one of the fragments C (we shall refer to it as C2) as well as the fragments G, H, K and L (Table III) the fragment G may be followed by the fragment H, L or C2. The fragments H and C2 are cleaved by R.EcoRI (see above) so that the R.HindIII fragment G can be followed only by the R.HindIII fragment C:, since otherwise the fragment H would not be cleaved by R.EcoRI as the molecular weight of the remaining part of the R.EcoRI fragment B (1.4 • 10 6) is somewhat higher than that of the R.HindIII fragment H. The R.HindIII fragment L was located TABLE
In
SECONDARY
CLEAVAGE
Cleaved fragments
OF INDIVIDUAL
Mol.
ADENOVIRUS
Cleavage products
TYPE 6 DNA FRAGMENTS
a
weights (X 1 0 - 6 )
R.HindlII
R.BgnI
A,CI,D,E,F,I,J, M + 2.95 G,K + 1.40 L + 0.7 and 0.64 0.63 and 0.41
A,B,C,E,I, + 1.5 F,tI,J,K + 0.35 1.1 and 0.4 N o cleavage
R.EcoRI
A B C D
16.5 3.65 1.56 1.05
R.BamHI
A B C D
8.5 "/.1 4.05 3.0
C2,G,H,K,L + 3.15 CI,F + 3.0 E + 1.8 and 0.25 I,J,M + 0.5 and 0.3
D,F,G,H,I,J,K B,E + 1.55 3.2 and 0.7 N o cleavage
R.HindIII
C2
2.14
--
J + 0.8 and 1.0
the m o l e c u l a r weights (X 1 0 - 6 ) o f t h e D N A fragments remaining after cleavage o f the original fragment w h i c h d o n o t b e l o n g t o a n y o f t h e i d e n t i f i e d fragments.
a The figttres denote
223 in the R.EcoRI fragment C {Table III) along with two DNA fragments which are the remainders of the fragments H and C2 {Table III). Such cleavage of the R.EcoRI fragment C is possible only for the following order of the R.HindIII fragments: H-L-C2-G-K (Fig. 5). The fragment H is followed b y the R.HindIII fragment B as shown b y the results of simultaneous cleavage with R.HindIII and R.EcoRI and with R.HindIII and R.BamHI, since only the R.HindIII fragment B is cleaved b y both enzymes (R.BamHI and R.EcoRI) and its molecular weight (3.18 • 106) is larger than the molecular weight of the remainder of the R.BamHI fragment A (3.05" 106). The R.BamHI fragment C is cleaved by R.HindIII into the R.HindIII fragment E (Table III) and two DNA fragments, one of which has the molecular weight 0.25 • 106 and the other 1.8 • 106. Fragment with the molecular weight 0.25 • 106 is a part of the R.HindIII fragment B since the latter is partially located in the R.BamHI fragment C. Hence, the R.HindIII fragment B is followed by the R.HindIII fragment E. The following order of the R.HindIII fragments has been determined: E-B-H-L-C2-G-K (Fig. 5). N o w let us consider the left side of the adenovirus type 6 DNA molecules. The R. BamHI fragment B is cleaved by R.HindIII into the fragment F, one of the fragments C (denoted as C1) and a fragment with a molecular weight 3.0" 106 (Table III). Since the second end fragment cleaved by R.HindIII is the fragment F (Fig. 5.1) it can be followed only by the fragment Cl. The latter should be followed by the fragment A because it is the only remaining R.HindIII fragment which can accomodate a DNA fragment with the molecular weight 3.0 • 106. Cleavage of the R.BamHI fragment C by R.HindIII produces the fragment E and two fragments (see above) one of which has the molecular weight 1.8 • 106 (Table III). The only remaining R.HindIII fragment which can contain this fragment is the fragment D. Thus, the partial R.HindIII cleavage map of the adenovirus type 6 DNA can be presented as follows: F-C~-A-I, J, M-D-E-B-H-LC2-G-K {Fig. 5). Cleavage of the R.BamHI fragment D by R.HindIII produced the fragments I, J and M and two fragments (we shall refer to them as X and Y, respectively) with the molecular weights 0.32 • 106 and 0.5 • 106 (Table III). The fragments X and Y are the remainders of the R.HindIII fragments A and D. The fragment X is a part of the R.HindIII fragment A because the sum of molecular weights of the remainders of the R.BamHI fragments B and D cleaved by R.HindIII (3.0 • 106 and 0.5 • 106, respectively) is in a good agreement with the molecular weight of the R.HindIII fragment A (Table III). Furthermore, Y is a part of the R.HindIII fragment D, since the molecular weight of the R.HindIII fragment D is in a good agreement with the sum of the molecular weights (1.8 • 106 and 0.3 • 106, respectively) found for the parts of the R.BamHI fragments C and D cleaved by R.HindIII (Table III). To determine the order of the fragments I, J and M the R.BamHI fragment D was partially cleaved by the enzyme R.HindIII. Fig. 3.9 (Table III) shows that the fragment X is associated with the fragment M and that the fragment Y is associated with the fragment J. Hence, the R.HindIII fragments are arranged in the R.BamHI fragment D in the following order: X(-M-I-J-)Y. Thus, we suggest the following order of the R.HindIII fragments in the adenovirus type 6 DNA: F-C~-A-M-I-J-D-E-B-H-L-C2-G-K (Fig. 5).
224
Cleavage of the adenovirus type 6 DNA by R.BglII and the order of the fragmen ts Complete cleavage of the adenovirus type 6 DNA by R.BglII gives rise to 11 fragments. Agarose gel electrophoresis shows nine of these fragments (Fig. 4.2). Two small fragments J and K are found in the agarose-acrylamide copolymer (Fig. 4.3). The adenovirus type 6 DNA was labelled with z2p in vitro
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225 (nick translation) and in vivo and cleaved by R.BglII. Gel electrophoresis and autoradiography of the labelled material have shown that all the cleaved fragments are present in the DNA in equimolar amounts. The fragments E and H were not found in gel after electrophoresis of the mixture of the R.BglII fragments produced by cleavage of the DNA-protein complex (Fig. 4.1, Table I). This means that t h e y are the end fragments. The products of simultaneous cleavage of the adenovirus type 6 DNA by R.BglII and R.EcoRI did not contain the R.BglII fragments D and G but contained the R.EcoRI fragment D (Fig. 4.5). This fact suggests that the R.BglII fragments D and G are located on the right side of the adenovirus type 6 DNA where the R.EcoRI fragments D, C and B are located. The products of simultaneous cleavage of the adenovirus type 6 DNA by R.BglII and R.BamHI included the R.BamHI fragment D but not the R.BglII fragments A and C (Fig. 4.7). Further, we analyzed individual R.BamHI and R.EcoRI fragments which we had mapped previously. Cleavage of the R.BamHI fragment B by R.BglII gave rise to the R.BglII fragments E and B and a DNA fragment with the molecular weight 1.55 • 106 (Table III). Since E is the end fragment, the R.BglII fragment B is located next to it. As R.BglII does not cleave the R.BamHI fragment D (see above) which is directly adjacent to the R.BamHI fragment B (Fig. 5) the smallest uncleared fragment should have the molecular weight 4.55 • 106 (3.0 • 106 + 1.55 • 106). Hence, the R.BglII fragment B is followed by the fragment A since it is the only fragment with an appropriate molecular weight (4.8 • 106). A comparison of the R.BglII fragments found in the R.EcoRI fragment A (A, B, C, E, I) and in the R.BamHI fragment A (D, F, G, H, I, J, K) suggests that the fragment I is located in the part where the R.EcoRI fragment A and the R.BamHI fragment A overlap (Table III), this means that the series of the R.BglII fragments E-B-A is followed by the fragments C and I (Fig. 5). The R.EcoRI fragment A cleaved by R.BglII gives rise to 5 R.BglII fragments and a fragment with the molecular weight 4.5 • 106 (Table III). The R.EcoRI fragment D adjacent to the R.EcoRI fragment A (Fig. 5) is not cleaved by R.BglII. Hence, the smallest molecular weight of the uncleaved fragment should be 2.55 • 106 (1.5 • 106 + 1.05 • 106). Only the R.BglII fragment D has a higher molecular weight (3.25-106) among the R.BglII fragments which remain unassigned. Hence, the R.BglII fragment I is followed by the fragment D. Since the R.EcoRI fragment B contains fragments F, H, J and K (Table III) the R.BglII fragment D can be followed only by the fragment G which is the only one among the unmapped R.BglII fragments that is cleaved by R.EcoRI (Fig. 4.5). Since the second end fragment of the adenovirus type 6 DNA cleaved by R.BglII is the H fragment (Fig. 4.1) the remaining three fragments to be located are the fragments F, I and K which are found in the R.EcoRI fragm e n t B and, hence, in the R.HindIII fragments C2 and G (Fig. 5). The R.HindIII fragment C2 was derived from the R.BamHI fragment A, labelled with 32p by means of nick translation and cleaved by R.BglII producing the fragment J and two additional DNA fragments (Table III). This can occur only if the R.BglII fragment G is followed by the fragment J, and the R.BglII fragments J and K are separated by the fragment F. Thus, the following order is
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suggested for the R.BglII fragments in the adenovirus type 6 DNA: E-B-A-C-ID-G-J-F-K-H (Fig. 5). Fig. 5 presents our physical maps of the adenovirus type 6 DNA for five enzymes: R. BamHI, R.EcoRI, R.SalI, R.BglII and R.HindIII. A comparison of our results with the physical maps for the DNAs of the group C adenoviruses (types 2 and 5) (Roberts, R., unpublished data) shows that the recognition sites for the above restriction enzymes on the DNAs of these adenoviruses have similar locations. However, there are certain differences, localized in definite parts (82--95%) of the genome map. This part of DNA, as one can deduce from the adenovirus functional map [30], probably codes for fiber protein, a major antigenic determinant responsible for the serotype specificity [31]. Very similar patterns of restriction sites in the left part (0--15%) of the genome of group C adenoviruses might be of interest. In adenovirus types 2 and 5 DNA
TABLE
IV
MOLECULAR WEIGHTS OF THE PRODUCTS R.BamHI FRAGMENT D BY R.HindIII
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Fragment order
I.--1.24 J.--0.77 X.--O.5 Y.--0.32 M.--0.046
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2.01 1.85 1.29 1.09 0.55
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227
this part comprises the gene(s) responsible for the transforming activity of the virus [32]. This is true also for adenovirus type 6, since R.HindIII fragment F is able to transform newborn rat kidney cells in culture (Naroditsky, B.S., unpublished results). The results obtained demonstrate a close structural similarity of adenovirus type 6, type 2 and type 5 genomes, although G + C content of adenovirus type 6 is somewhat different. This allows extrapolation of adenovirus types 2 and 5 functional mapping data to type 6. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Smith, H.O. and Nathans, D. (1973) J. Mol. Biol. 81, 419--423 Huebner, R.J. (1967) Perspect. Virol. 5, pp. 147--198 Gilden, R.V., Kern, I. and Freeman, A.S. (1968) Nature 2 1 9 , 5 1 7 - - 5 1 8 Green, M., Pina, M., Kimes, R., Weisink, P.C., McHattie, L.A. and Thomas, C.A. (1967) Proc. Natl. Acad. Sci. U.S.A. 57, 1302--1309 DanieU, E. (1976) J. Vixol. 1 9 , 6 8 5 - - 7 0 8 Mulder, C., Arrand, J.R., DeUius, H., Keller, W., Petterson, U., Roberts, R.J. and Sharp, P.A. (1974) Cold Spring Harbor Syrup. Quant. Biol. 3 9 , 3 9 7 - - 4 0 0 Naroditsky, B.S., Karamov, E.V., Zavizion, B.A., Tikchonenko, T.I. and Dreisin, R.S. (1976) Voprosy Virnsol. (U.S.S.R.) 5, 539--544 Naroditsky, B.S., Felicina, T.I., Karamov, E.V. and Tikchonenko, T.I. (1977) Dokladi Akad. Nauk. (U.S.S.R.) 2 3 7 , 2 2 6 - - 2 3 2 Forsblom, S., Rigler, R., Ehunbery, M., Petterson, U. and Phflipson, L. (1976) Nucl. Acid. Res. 3, 3255 --3269 Dreisin, R.S., Zolotarskaya, E.E. (1966) Voprosy Virusol. (U.S.S.R.) 6 , 6 8 9 - - 6 9 5 Green, M. and Pina, M. (1963) Virology 20, 199--207 Maisel, I.V., White, D.O., Scharff, M.D. (1968) Virology 3 6 , 1 1 5 - - 1 2 5 Bello, Z.l. and Grinsberg, H.S. ( 1 9 6 9 ) J . Vixol. 3, 106--119 Ioshimori, R.H. (1972) Ph.D. Thesis. University of California, San Francisco Medical Center, San Francisco Naroditsky, B.S., Zavizion, B.A., Kaxamov, E.V. and Tikchonenko, T.I. (1978) Nucl. Acid Res. 5, 999--1012 Bickle, A. and Pirotta, V. (1977) Nucl. Acid Res. 4, 2131--2140 Wilson, G.A. and Yaung, F.S. (1975) J. Mol. Biol. 97, 123--125 Sharp, P.A., Sagden, B. and Sambrook0 J. (1973) Biochem. 12, 3055--3063 Helling, R.B., Goodman, H.W. and Boyer, H.W. (1974) J. Vixol. 14, 1235--1244 Thomas, M. and Davis, R.W. (1975) J. Mol. Biol. 91, 315--328 Edgell, M.N., Hutchison, C.A. III and Scloir, M. (1972) J. Virol. 9, 574--782 Davis, R.W., Simon, M.N. and Davidson, N. (1971) Methods Enzymol. 2 1 , 4 1 3 - - 4 2 8 Delins, H., Westful, H. and Axeirod, N. (1973) J. Mol. Biol. 74, 677--678 Palleyblank, D.E., Shure, M., Tong, D., Vinograd, J. and Verberg, H.F. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4 2 8 0 - - 4 2 8 4 Sambrook, J., Botchan, M., Gallemor, P.M., Ozanne, B., Petterson, U., WiUiamson, J. and Sharp, P. (1974) Cold Spring Harbor Syrup. Quant. Biol. 70, 57--71 Maniatis, T., Lefbrey, A., Kleid, D.C. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1184--1188 Robinson, A.I., Yonghusband, H.B. and Bellett, A.I.D. (1973) Virology 56, 54--69 Sharp, P.A., More, C. and Haverty, I.L. (1976) Virology 75, 442--456 Lai, C.L. and Nathans, D. (1974) J. Mol. Biol. 83, 179--193 Lewis, J.B., Atkins, J.F., Anderson, C.W., Baum, P.R. and Gesteland, R.F. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1 344--1348 Phflipson, L., Petterson, U. and Lindberg, U. (1976) Mol. Biol. Adenoviruses, 14, 1--115 Van der Eb, A.G. and Honweling, A. (1977) Gene 2, 133--146
Biochimica et Biophysica Acta, 606 (1980) 214--227
© Elsevier/North-Holland Biomedical Press
BBA 99589 ANALYSIS OF DNA FROM HUMAN ADENOVIRUS TYPE 6 WITH RESTRICTION ENDONUCLEASES HindIII, BglIII and BamHI
B.S. NARODITSKY a, T.I. KALININA a, E.Z. GOLDBERG a, A.S. BOROVIK b , E.V. KARAMOV a and T.I. TIKCHONENKO a,. a D.I. Ivanovsky Institute o f Virology, the USSR Academy o f Medical Sciences, Gamaleya str. 16, Moscow 123098 and b Molecular Genetics Institute, the USSR Academy o f Sciences, Moscow (U.S.S.R.)
(Received January 15th, 1979) (Revised manuscript received July 4th, 1979) Key words: DNA map; Restriction endonuclease; (Adenovirus)
Summary The genome of the type 6 h u m a n adenovirus has three restriction sites for R.BamHI, thirteen for R.HindIII and ten for R.BglII. The terminal fragments of DNA cleaved by each of the enzymes have been determined by means of terminal nucleotidyl transferase and by analysis of the DNA-terminal protein complex. The sequence of the cleaved fragments has been determined by partial cleavage of DNA, simultaneous digestion of DNA with various combinations of enzymes and secondary digestion of individual isolated fragments with other enzymes. The following order of the cleaved fragments in the adenovirus t y p e 6 genome has been found (the figures in brackets are the weights in megadaltons): for R.BamHI-B(7,1)-D(3.0)-C(4.05)-A(8.5); for R.HindIII-F(1.7)Cl (2.14)-A(3.44)-M(0.046)-I(1.24)-J(0.77)-D(2.1)-E(1.96)-B(3.18)-H(1.36)L(0.18)-C2(2.14)-G(1.44)-K(0.16); for R.BglII-E(2.07)-B(3.5S)-A(4.8)-C(3.36)I(0.7S)-D(3.25)-G(1.37)-J(0.21)-F(1.85)-K(0.17)-H(0.94).
Introduction The h u m a n adenovirus of the type 6 belongs to the group of nononcogenic h u m a n adenoviruses [2] but it can induce in vitro transformation of some * TO w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . A b b r e v i a t i o n s : R . E c o R I , R . B a m H I , R . H i n d l I I a n d R . B g l I I - r e s t r i c t i o n e n d o n u e l e a s e s f r o m Escherichia coli R Y 13, Baeilus amyloliquefaeiens, Haemophilus inl'luenzae R d a n d Bacillus globigii, r e s p e c t i v e l y . T h e r e s t r i c t i o n e n d o n u c l e a s e s are c l a s s i f i e d a c c o r d i n g t o t h e n o m e n c l a t u r e [ 1 ] . 3 2 p - d N T P , d e o x y t r i p h o s phage labelled w i t h r a d i o a c t i v e p h o s p h o r u s at t h e a p o s i t i o n .
215
cells [3]. The adenovirus type 6 genome is a linear double helical DNA molecule with the molecular weight about 23 • 106 (see below). G + C content in adenovirus type 6 DNA is 52.6% (Tikchonenko, T.I., unpublished results) which is lower than the value {56--60%) that should be revealed in adeno DNA in accordance with Green rule [4]. In contrast to other adenoviruses [5,6] of this group, t o o few studies have been made concerning the physicochemical properties of the adenovirus type 6 DNA and its cleavage with restriction endonucleases. We have reported earlier [7,8] on the number of recognition sites and the sequence of cleaved fragments in adenovirus type 6 DNA for R.EcoRI and R.SalI. Later our results for the R.EcoRI fragments have been confirmed b y Forsblom [9], who used another technique for mapping the DNA fragments. This paper presents the results of mapping of the adenovirus t y p e 6 DNA fragments cleaved with three other restriction enzymes, namely R.BamHI, R.HindIII and R.BglII. Materials and Methods
Cell culture, virus growth and DNA extraction. The adenovirus type 6 was propagated in HeLa cells of the Bristol line [10]. The virus was extracted from the infected cells by freezing and thawing out. The extracted virus was treated with Freon-113 and concentrated by centrifugation on the CsC1 cushion (p = 1.43 g/cm 3) according to the procedure [11]. The concentrated viral suspension was further purified by t w o cycles of equilibrium centrifugation in CsC1 density gradient [12]. Electron microscopy was used to control the purity of the viral preparation. The viral DNA was isolated according to the modified p r o c e d u r e [13]. Source o f enzymes. The endonuclease R.EcoRI was isolated from the E. coli RY-13. The restriction enzyme was purified according to the modified procedure [14]. The enzyme R.BamHI was isolated according to the procedure [15]. The enzyme R.HindIII was supplied b y Miles (U.S.A.) and R.BglII was isolated according to the procedure [16]. The media for the enzyme reactions were: for R.EcoRI: 70 mM Tris-HC1, pH 7.5/50 mM NaC1/10 mM MgC12/7 mM 2-mercaptoethanol [14]; for R.BamHI: 6 mM Tris-HC1, pH 7.4/6 mM MgC12/ 6 mM 2-mercaptoethanol [17]; and for R.HindIII and R.BglII: 10 mM TrisHC1, pH 7.5/6.0 mM MgC12/10 mM KC1/6 mM 2-mercaptoethanol [6]. The temperature was 37°C, the time of incubation was 15--90 min, the volume of the reaction mixture was 20--50 pl, the DNA content was 0.5--2 pg and the a m o u n t of enzyme varied from 0.5 to 5 ~1. To produce partial cleavage the viral DNA was incubated either with diluted enzyme or in a medium with a higher ionic strength at 30°C. The enzyme reaction was stoped b y adding EDTA or incubating the mixture for 5 min at 65°C. Electrophoretic analysis. The cleaved fragments were analyzed by agarose gel electrophoresis [18,19] in tubes or in 3--5 mm slabs of agarose gel (Bio-Rad, U.S.A.). The gel was stained in 1/~g/ml solution of ethidium bromide (Calbiochem, U.S.A.) for 20 min and ultraviolet photographs were used to detect the positions of the DNA fragments. The internal standard for calculating the molecular weights of the cleaved fragments was the R.EcoRI fragments of the phage ~ DNA, the molecular weights of which were taken from Ref. 20. In electrophoresis of labelled material the gels after staining with ethidium
216 bromide were either used for exposing high-sensitivity X-ray film or cut into 1-mm disks which were put into vials for measuring radioactivity, dissolved with the Soluen universal solvent (0.4 ml per disk) and stored for 2 h at 60°C. Then 10 ml of toluene scintillator were added and the activity was measured with an SL-30 scintillation counter (Intertechnik, France). Analysis of the adenovirus type 6 DNA fragments in the agarose-polyacrylamide copolymer was conducted according to the procedure [21]. Electron microscopy. The DNA preparations were made according to the modified formamide procedure [22]. 25 ~l of c y t o c h r o m e c, cleaved with 10 t~g/ml BrCn [23], and 25 ~1 of 10 pg/ml DNA were added to 50 t~l of the buffer solution containing 0.01 M Na3EDTA, 0 . 0 4 M phosphate buffer (pH 7.0) and 90% formamide (Merck, F.R.G.). Deionized water was used as the hypophase. The protein film on the water surface was applied to a copper grid coated with a thin carbon film. The specimen was shadowed with platinum and photographed in a JEM-7 electron microscope. The magnification was calibrated by the replica of the diffraction grating. Elution o f DNA from the agarose gel. Following electrophoresis of DNA the agarose gel was stained with ethidium bromide and the gel band containing a given DNA fragment was cut off, frozen, placed into a centrifuge test-tube and centrifuged in a SW-50.1 rotor for 1 h at 30°C and 35 000 rev./min in an L-5-50 centrifuge (Beckman, U.S.A.). The supernatant was collected and deproteinized 2 or 3 times, and the DNA was precipitated with ethanol [24]. Preparation o f the 32p-labelled adenovirus type 6 DNA. The 32p-labelled DNA was obtained either in vivo according to the procedure [25] with the specific activity of 2 • 10 s cpm/~g or by the nick-translation reaction [26]. In the latter case the specific activity of the preparation was 1 • 106--1.2 • 106 cpm/~g. The 3:P-labelled dATP (Amersham, U.K.) was used as the labelled precursor. Preparation o f the adenovirus type 6 DNA-terminal protein complex. The DNA-protein complex was prepared from purified viral suspension in the presence of 4 M guanidine chloride and the complex was purified by CsC1 equilibrium centrifugation in the presence of guanidine chloride [27 ]. Results and Discussion
Cleavage and mapping o f the R.BamHI fragments o f the adenovirus type 6 DNA Complete cleavage of the adenovirus type 6 DNA by R.BamHI gives rise to four fragments, A, B, C and D (Fig. 1.5). Table I presents the molecular weights of the R.BamHI fragments. The direct electron microscopy determinations of the amounts and lengths of the R.BamHI fragments also reveal the presence of four types of fragment. The histogram in Fig. 2 shows that all the fragments are present in equimolar amounts. The molecular weights of the fragments calculated from the length measurements are in a good agreement with the electrophoretic results; the total molecular weight of the R.BamHI fragments of the adenovirus type 6 DNA (22.9 • 106) agrees with the electron microscopy result (23.1 • 106) for the native adenovirus type 6 DNA and with the total molecular weights of the R.EcoRI fragments (22.76" 106) and the R.SalI fragments (22.85 • 106) of the adenovirus type 6 DNA [7,8].
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7
8
Fig. 1. Cleavage of t h e a d e n o v i r u s t y p e 6 D N A b y R . B a m H I a n d a m i x t u r e of R . B a m H I a n d R . E e o R I . Capital l e t t e r s d e n o t e t h e p r o d u c t s of c o m p l e t e cleavage. R o m a n n u m e r a l s d e n o t e t h e p r o d u c t s of p a r t i a l cleavage. T h e D N A f r a g m e n t s w e r e a n a l y z e d in 0.7% agarose slab gel at 1.5 V / c m for 1 8 - - 2 2 h, or in 1% agarose t u b e s . 1. D N A o p r o t e i n c o m p l e x t r e a t e d w i t h R . B a m H I a n d t h e n i n c u b a t e d w i t h 10/~g of p r o n a s e at 3 7 ° C f o r 15 rain. 2. D N A - p r o t e i n c o m p l e x t r e a t e d w i t h R . B a m H I . 3. A d e n o v i r u s t y p e 6 D N A d i g e s t e d u n d e r t h e p a r t i a l cleavage c o n d i t i o n s (0.5/~1 R . B a m H I , 10 rain, 3 2 ° C , 0.3 M NaC1). 4. P r o d u c t s o f s e c o n d azy R . B a m H I cleavage o f the D N A f r a g m e n t s isolated f r o m the b a n d w i t h the m o l e c u l a r w e i g h t 7 • 106. 5. A d e n o v i r u s t y p e 6 D N A digested u n d e r the c o n d i t i o n s of c o m p l e t e cleavage (1 /~1 R . B a m H I , 15 rain, 3 7 ° C ) . 6. T h e R . E c o R I f r a g m e n t s o f the a d e n o v i r u s t y p e 6 D N A . 7. A d e n o v i r u s t y p e 6 D N A c l e a v e d s i m u l t a n e o u s l y b y R . E c o R I a n d R . B a m H I . T h e cleavage c o n d i t i o n s axe t h e s a m e as f o r cleavage b y R . E c o R I . 8. T h e R . B a m H I f r a g m e n t s o f a d e n o v i r u s t y p e 6 D N A .
The order of the R.BamHI fragments of the adenovirus type 6 DNA was determined from the results of partial cleavage of DNA and by identification of the terminal fragments using the analysis of the DNA-terminal protein complex. The terminal fragment covalently linked protein produced in cleavage of the complex does not enter agarose gel [28]. The fragments A and B were not found in the agarose gel after electrophoretic separation of the restriction mixture, while the fragments C and D had the typical mobility in the gel (Fig.
I
a b c d
D E F G H I J K L M
A B C
8.4 7.8 6.4 6.15 5.16 3.35 2.41 0.73 --
29.6 d
16.1 13.9
Distribution of 32p (%)
R.HindIII
MOLECULAR
_+ 0 . 1 5 _+ 0 . 1 2 ± 0.14 X 2
2.1 _+ 0 . 0 9 1.96 ± 0.08 1 . 7 _+ 0 . 1 1 c 1.44 ± 0.06 1 . 3 6 +_ 0 . 0 7 1.24 + 0.09 0 . 7 7 _+ 0 . 0 5 0 . 6 _+ 0 . 0 6 c 0.18 ± 0.01 0.046 ± 0.004 22.4
3.44 3.18 2.14
OF
THE
15.36 14.20 9.55 X 2 (C 1 + C 2 ) 9.38 8.75 7.59 6.43 6.07 5.54 3.44 2.68 0.80 0.21 Z 100%
Percentage of the genome
WEIGHTS
Molecular weight a (X 1 0 - 5 )
fragments
AND
OF
9.72 7.9 6.17 4.51 3.02 0.89 0.66
30.35 d
20.33 16.4
Distribution of 32p (%)
THE
_+ 0 . 0 7 + 0.08 +- 0 . 1 ± 0.12 _+ 0 . 1 1 ± 0.09 _+ 0 . 0 0 5 _+ 0 . 0 0 5
Z 22.37
3.25 2.07 1.85 1.37 0.94 0.78 0.21 0.17
4.8 + 0.2 3.58 + 0.12 3.36 + 0.06
TYPE
~ 100%
14.53 9.26 c 8.72 6.13 4.2 c 3.49 0.98 0.76
21.47 16.01 15.03
Percentage of the genome
ADENOVIRUS
Molecular weight a (X 1 0 - 5 )
R.BglII fragments
FRAGMENTS
2~ 2 2 . 9
2.95
8.87 7.05 4.03
Electron microscopic mol.wt, b (X 1 0 - 6 )
_+ 0 . 1 2
C and D.
~ 22.65
3.0
8.5 ÷ 0.25 7 . 1 _+ 0 . 2 1 4 . 0 5 +_ 0 . 1 5
Molecular weight (X 1 0 -6 )
BY R.HindIII,
fragments
CLEAVED
R.BamHI
6 DNA
The values are means from 7--11 experiments. Measttrements of electrophoretic mobility in agarose and agarose-acrylamide gel. Col EI DNA was used as the internal standard. The end fragments were determined from analysis of the DNA-protein complex. S i n g l e r a d i o a c t i v i t y p e a k s w e r e d e t e c t e d f o r t h e b a n d s c o r r e s p o n d i n g t o t h e R . H i n d I I I f r a g m e n t s C 1 , C2 a n d D a n d t h e R . B g l I l f r a g m e n t s
Fragments
THE NUMBERS R.BamHI
TABLE
13.25
37.53c 31.35 c 17.88
Percentage of the genome
R.BglII AND
b~
219 D
C
N 30 3 kJ o
E 20
B
E
3 Z
10 A
Ifl.I 1
11 2
Contour
oA
!
i
3
4
pM
length
Fig. 2. H i s t o g r a m o f l e n g t h m e a s u r e m e n t s (/~m) f o r t h e R . B a m H I f r a g m e n t s o f a d e n o v i r u s t y p e 6 D N A . P e r c e n t a g e s o f t h e f r a g m e n t s in r e l a t i o n t o t h e t o t a l n u m b e r o f m e a s u r e d m o l e c u l e s : A , 1 9 ; B, 2 2 ; C, 23; a n d D, 2 5 .
1.2). When before electrophoretic analysis the same mixture of fragments was treated with pronase, all four R.BamHI fragments were found in the gel (Fig. 1.1). The 3'-labelling of intact DNA with the use of terminal nucleotidyltransferase was chosen as an independent method for the determination of the end fragments. The fragmentation of the DNA with BamHI and the subsequent fractionation of the hydrolyzate in agarose gel have shown that A and B are the end fragments. The determine the order of the internal R.BamHI fragments in the adenovirus type 6 DNA the optimum conditions were selected for partial cleavage of the DNA. The products of partial cleavage contained, apart from the four fragments (A, B, C and D) produced by complete cleavage, additional fragments denoted as I, II, III, IV and V in the order of decreasing molecular weight (Figs. 1.3, 1.4 and Table II). The molecular weights of these fragments correspond to the sums of weights of the fragments of complete cleavage: AC, ACD, BCD, BD and CD (Table II). To detect the fragment V which has the same molecular weight as fragment B, DNA was eluted from the gel band where the nucleic acid with molecular weight 7" 10 6 was located. After secondary cleavage of this DNA by R.BamHI and separation of the cleaved fragments in the agarose gel, the fragments C and D were found apart from the fragment B (Fig. 1.4). The good agreement between the predicted values and the measured molecular weights of the partial cleavage products (Table II) makes it possible to determine the fragment composition of the partial cleavage products and shows that the fragment D is between the fragments C and B and the fragment C is associated with the fragment A. Thus, the above results on the
220 TABLE
II
MOLECULAR WEIGHTS OF THE TIAL CLEAVAGE WITH R.BamHI
ADENOVIRUS
TYPE
6 DNA
FRAGMENTS
PRODUCED
BY PAR-
The weights are means of the results of six individual experiments. Fragments
Expected tool. weight ( × 1 0 -6 )
I II Ill IV
V
Measured tool. weight ( X I O -5 )
Fragment composition
Overlapping order
15.55
15.8 + 0.3
A,C,D
ACD
14.15
13.9 ÷ 0.2
B,C,D
CDB
12.55 10.10
12.8 + 0.2 9.9 • 0 . I
A,C B,D
AC DB
7.05
7.0 + 0.1
C,D
CD
products of cleavage of the adenovirus type 6 DNA by R.BamHI show clearly the following order of the cleaved fragments in the DNA: A-C-D-B (Fig. 5).
The mutual arrangement of the R.EcoRI and R.BamHI fragments To determine the mutual arrangement of the R.EcoRI and R.BamHI fragments, the adenovirus type 6 DNA was simultaneously treated with R.EcoRI and R.BamHI. The products of the reaction did not contain the R.EcoRI fragment A and the R.BamHI fragment A; the products B, C and D of both enzymes were retained and an additional fragment with the molecular weight 2.6 • 106 was found (Fig. 1.7). The results show that the R.EcoRI fragments B, C and D comprise the part of the adenovirus type 6 DNA which corresponds to the R.BamHI fragment A and the R.BamHI fragments B, C and D are located in the part of the adenovirus type 6 DNA which corresponds to the R.EcoRI fragment A. These results suggest that the restriction sites for R.EcoRI and R.BamHI are at the opposite ends of the DNA molecule (Fig. 5). Cleavage o f the adenovirus type 6 DNA by R.HindIII and the order o f the R.HindlII fragments The enzyme R.HindIII cleaves the adenovirus type 6 DNA into 14 fragments (Table I, Fig. 3). The number of fragments was determined by analysis of 3:p-labelled DNA labelled in vivo or in vitro (nick translation). The fragments from A to K were found in the agarose gel (Fig. 3.2) and the fragment L in the agarose-acrylamide copolymer (Fig. 3.3). For detection of fragment M it was necessary to use highly labelled 32p_ labelled DNA preparation and to conduct electrophoretic separation of restriction fragments in 5% polyacrylamide gel with subsequent autoradiography (Fig. 3.4). Quantitative analysis of the distribution of 32p in the gel after separation of the R.HindIII fragments demonstrated that the third largest band contains two DNA fragments with the same molecular weight (Table I) which we denoted as C~ and C2. The fragment D has a molecular weight which is very close to that of the fragments C, and C2 and it can be separated from them only in the 1.4% agarose gel.
221
-~
-ff +
"1-
t
+
E
I1J
rn
-~
o~
E
-r
0
,'Y
E
oLI
13
I
rn
u.I
4-
4"
'13 E
e-.
13 ('-
..~ 4.-
E- E ~ _ C3 "1-
II A B
3~C~EfC2 F 3H I X
J K 1 2
3
4
5
6
7
Fig. 3. E l e c t r o p h o r e s i s o f t h e R . H i n d I I I f r a g m e n t s o f t h e a d e n o v i r u s t y p e 6 D N A . Capital l e t t e r s d e n o t e the f r a g m e n t s o f c o m p l e t e cleavage. R o m a n n u m e r a l s d e n o t e t h e p r o d u c t s o f p a r t i a l cleavage. T h e D N A f r a g m e n t s w e r e s e p a r a t e d in 1% or 1.4% a g a r o s e slab gel at 1.5 V / c m f o r 2 0 - - 2 2 h a t 4°C (1, 2, 5), in a g a r o s e - a c r y l a m i d e c o p o l y m e r ( 0 . 5 % agarose + 2.5% a c r y l a m i d e ) at 4 V / c m f o r 2 3 - - 2 4 h [ 3 ] a n d in 5% p o l y a c r y l a m i d e slab gel [ 4 ] . 1. D N A - p r o t e i n c o m p l e x t r e a t e d b y R . H i n d I I I . 2, 5. A d e n o v i r u s t y p e 6 D N A ( 0 . 7 - - 1 ~zg) t r e a t e d b y R . H i n d I I I ( 1 - - 2 /~1) at 3 7 ° C f o r 1 h. 3. A d e n o v i r u s t y p e 6 D N A (5/~g) t r e a t e d b y R . H i n d I I I (4 ~ul) a t 3 7 ° C f o r 2 h. 4. T h e R . B a m H I f r a g m e n t D labelled w i t h 3 2 p in v i t r o ( n i c k t r a n s l a t i o n ) a n d t r e a t e d b y R . H i n d I I I ( a u t o r a d i o g r a p h y ) . 6. S i m u l t a n e o u s cleavage o f t h e a d c n o v i r u s t y p e 6 D N A b y R . H i n d l I I a n d R . B a m H I u n d e r the s t a n d a r d c o n d i t i o n s of R . H i n d I I I cleavage. 7. S i m u l t a n e o u s cleavage of the adenovirus type 6 DNA by R.HindllI and R.EcoRI under the standard conditions of R.HindIII cleavage. 8. T h e R . B a m H I f r a g m e n t D (0.4 #g) t r e a t e d b y R . H i n d I I I u n d e r t h e c o n d i t i o n s o f p a r t i a l cleavage (1 ~1, 3 7 ° C , 1 h).
222
Analysis of the R.HindIII fragments produced by cleavage of the DNAprotein complex in the agarose gel demonstrated the absence of the fragments F and K (Fig. 3.1, Table I) suggesting that they are the end fragments (see above). Various techniques were used for determination of the order of the R.HindIII fragments in the adenovirus type 6 DNA. Firstly, the adenovirus t y p e 6 DNA was simultaneously treated with R.HindIII and R.EcoRI, and with R.HindIII and R.BamHI. When the adenovirus type 6 DNA was treated with R.HindIII and R.EcoRI (Fig. 3.7) all the R.EcoRI fragments and B, one of the C fragments and H R.HindIII fragments disappeared in the mixture. Treatment of the adenovirus type 6 DNA with R.HindIII and R.BamHI led to disappearance of all the R.BamHI fragments and the A, B and D R.HindIII fragments (Fig. 3.5). Since we have mapped the R.EcoRI [7] and R.BamHI fragments (Fig. 5) it may be suggested that the R.HindIII fragments B, H and C are in the right half of the genome and the fragments A and D are in the left half of the molecule. Here we refer to the region of the adenovirus type 6 DNA containing the R.EcoRI fragment A as the left part of the molecule. Since the number of the R.HindIII fragments is relatively large we analyzed the individual adenovirus type 6 DNA fragments produced by R.EcoRI and R.BamHI for which the order of the fragments is known. Cleavage of the R.EcoRI fragment B with R.HindIII gave rise to the fragments G and K and a fragment with the molecular weight 1.4 • 10 6 (Table II). Since the fragment K is the end fragment, the fragment G is adjacent to it. Since the products of cleavage of the R.BamHI fragment A with R.HindIII contained one of the fragments C (we shall refer to it as C2) as well as the fragments G, H, K and L (Table III) the fragment G may be followed by the fragment H, L or C2. The fragments H and C2 are cleaved by R.EcoRI (see above) so that the R.HindIII fragment G can be followed only by the R.HindIII fragment C:, since otherwise the fragment H would not be cleaved by R.EcoRI as the molecular weight of the remaining part of the R.EcoRI fragment B (1.4 • 10 6) is somewhat higher than that of the R.HindIII fragment H. The R.HindIII fragment L was located TABLE
In
SECONDARY
CLEAVAGE
Cleaved fragments
OF INDIVIDUAL
Mol.
ADENOVIRUS
Cleavage products
TYPE 6 DNA FRAGMENTS
a
weights (X 1 0 - 6 )
R.HindlII
R.BgnI
A,CI,D,E,F,I,J, M + 2.95 G,K + 1.40 L + 0.7 and 0.64 0.63 and 0.41
A,B,C,E,I, + 1.5 F,tI,J,K + 0.35 1.1 and 0.4 N o cleavage
R.EcoRI
A B C D
16.5 3.65 1.56 1.05
R.BamHI
A B C D
8.5 "/.1 4.05 3.0
C2,G,H,K,L + 3.15 CI,F + 3.0 E + 1.8 and 0.25 I,J,M + 0.5 and 0.3
D,F,G,H,I,J,K B,E + 1.55 3.2 and 0.7 N o cleavage
R.HindIII
C2
2.14
--
J + 0.8 and 1.0
the m o l e c u l a r weights (X 1 0 - 6 ) o f t h e D N A fragments remaining after cleavage o f the original fragment w h i c h d o n o t b e l o n g t o a n y o f t h e i d e n t i f i e d fragments.
a The figttres denote
223 in the R.EcoRI fragment C {Table III) along with two DNA fragments which are the remainders of the fragments H and C2 {Table III). Such cleavage of the R.EcoRI fragment C is possible only for the following order of the R.HindIII fragments: H-L-C2-G-K (Fig. 5). The fragment H is followed b y the R.HindIII fragment B as shown b y the results of simultaneous cleavage with R.HindIII and R.EcoRI and with R.HindIII and R.BamHI, since only the R.HindIII fragment B is cleaved b y both enzymes (R.BamHI and R.EcoRI) and its molecular weight (3.18 • 106) is larger than the molecular weight of the remainder of the R.BamHI fragment A (3.05" 106). The R.BamHI fragment C is cleaved by R.HindIII into the R.HindIII fragment E (Table III) and two DNA fragments, one of which has the molecular weight 0.25 • 106 and the other 1.8 • 106. Fragment with the molecular weight 0.25 • 106 is a part of the R.HindIII fragment B since the latter is partially located in the R.BamHI fragment C. Hence, the R.HindIII fragment B is followed by the R.HindIII fragment E. The following order of the R.HindIII fragments has been determined: E-B-H-L-C2-G-K (Fig. 5). N o w let us consider the left side of the adenovirus type 6 DNA molecules. The R. BamHI fragment B is cleaved by R.HindIII into the fragment F, one of the fragments C (denoted as C1) and a fragment with a molecular weight 3.0" 106 (Table III). Since the second end fragment cleaved by R.HindIII is the fragment F (Fig. 5.1) it can be followed only by the fragment Cl. The latter should be followed by the fragment A because it is the only remaining R.HindIII fragment which can accomodate a DNA fragment with the molecular weight 3.0 • 106. Cleavage of the R.BamHI fragment C by R.HindIII produces the fragment E and two fragments (see above) one of which has the molecular weight 1.8 • 106 (Table III). The only remaining R.HindIII fragment which can contain this fragment is the fragment D. Thus, the partial R.HindIII cleavage map of the adenovirus type 6 DNA can be presented as follows: F-C~-A-I, J, M-D-E-B-H-LC2-G-K {Fig. 5). Cleavage of the R.BamHI fragment D by R.HindIII produced the fragments I, J and M and two fragments (we shall refer to them as X and Y, respectively) with the molecular weights 0.32 • 106 and 0.5 • 106 (Table III). The fragments X and Y are the remainders of the R.HindIII fragments A and D. The fragment X is a part of the R.HindIII fragment A because the sum of molecular weights of the remainders of the R.BamHI fragments B and D cleaved by R.HindIII (3.0 • 106 and 0.5 • 106, respectively) is in a good agreement with the molecular weight of the R.HindIII fragment A (Table III). Furthermore, Y is a part of the R.HindIII fragment D, since the molecular weight of the R.HindIII fragment D is in a good agreement with the sum of the molecular weights (1.8 • 106 and 0.3 • 106, respectively) found for the parts of the R.BamHI fragments C and D cleaved by R.HindIII (Table III). To determine the order of the fragments I, J and M the R.BamHI fragment D was partially cleaved by the enzyme R.HindIII. Fig. 3.9 (Table III) shows that the fragment X is associated with the fragment M and that the fragment Y is associated with the fragment J. Hence, the R.HindIII fragments are arranged in the R.BamHI fragment D in the following order: X(-M-I-J-)Y. Thus, we suggest the following order of the R.HindIII fragments in the adenovirus type 6 DNA: F-C~-A-M-I-J-D-E-B-H-L-C2-G-K (Fig. 5).
224
Cleavage of the adenovirus type 6 DNA by R.BglII and the order of the fragmen ts Complete cleavage of the adenovirus type 6 DNA by R.BglII gives rise to 11 fragments. Agarose gel electrophoresis shows nine of these fragments (Fig. 4.2). Two small fragments J and K are found in the agarose-acrylamide copolymer (Fig. 4.3). The adenovirus type 6 DNA was labelled with z2p in vitro
I=1
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u .c
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u
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o
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z
I
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t~
~
~
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>
m + ~
I=:1
~
03
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6
7
A B DC
E F G I H K I
1
2
3
4
5
Fig. 4. E l e c t r o p h o r e s i s o f t h e R.BglII cleavage p r o d u c t s of a d e n o v i r u s t y p e 6 D N A . Capital letters d e n o t e the p r o d u c t s o f c o m p l e t e cleavage. E l e c t r o p h o r e s i s in slab gel of 0.8 a n d 1% agarose at 1.5 V / c m f o r 1 8 - 2 0 h (1, 2, 5) a n d in 0.5% a g a r o s e - 2 . 5 % a c r y l a m i d e c o p o l y m e r at 4 V / c m at 23 h [ 3 , 4 ] . 1. D N A - p r o t e i n c o m p l e x t r e a t e d b y R.BglII. 2, 6 A d e n o v i r u s t y p e 6 D N A ( 0 . 7 - - 1 pg) t r e a t e d b y R . B g l I l ( 4 - - 6 ~l) at 3 7 ° C for 2 h . 3. A d e n o v i r u s t y p e 6 D N A (4 btg) t r e a t e d b y R.BglII ( 1 5 btl) at 3 7 ° C for 2 h . 4. M a r k e r SV-40 D N A (2/~g) t r e a t e d b y R . H i n d I I l (2 btl) at 3 7 ° C for 2 h [ 2 9 ] . 5. S i m u l t a n e o u s cleavage of the a d e n o v i r u s t y p e 6 D N A b y R.BglII a n d R . E c o R I u n d e r the s t a n d a r d c o n d i t i o n s for R.BgllI cleavage. 7. S i m u l t a n e o u s cleavage of the a d e n o v i r u s t y p e 6 D N A b y R.BgllI a n d R . B a m H I u n d e r t h e s t a n d a r d c o n d i t i o n s for R . B a m H I cleavage.
225 (nick translation) and in vivo and cleaved by R.BglII. Gel electrophoresis and autoradiography of the labelled material have shown that all the cleaved fragments are present in the DNA in equimolar amounts. The fragments E and H were not found in gel after electrophoresis of the mixture of the R.BglII fragments produced by cleavage of the DNA-protein complex (Fig. 4.1, Table I). This means that t h e y are the end fragments. The products of simultaneous cleavage of the adenovirus type 6 DNA by R.BglII and R.EcoRI did not contain the R.BglII fragments D and G but contained the R.EcoRI fragment D (Fig. 4.5). This fact suggests that the R.BglII fragments D and G are located on the right side of the adenovirus type 6 DNA where the R.EcoRI fragments D, C and B are located. The products of simultaneous cleavage of the adenovirus type 6 DNA by R.BglII and R.BamHI included the R.BamHI fragment D but not the R.BglII fragments A and C (Fig. 4.7). Further, we analyzed individual R.BamHI and R.EcoRI fragments which we had mapped previously. Cleavage of the R.BamHI fragment B by R.BglII gave rise to the R.BglII fragments E and B and a DNA fragment with the molecular weight 1.55 • 106 (Table III). Since E is the end fragment, the R.BglII fragment B is located next to it. As R.BglII does not cleave the R.BamHI fragment D (see above) which is directly adjacent to the R.BamHI fragment B (Fig. 5) the smallest uncleared fragment should have the molecular weight 4.55 • 106 (3.0 • 106 + 1.55 • 106). Hence, the R.BglII fragment B is followed by the fragment A since it is the only fragment with an appropriate molecular weight (4.8 • 106). A comparison of the R.BglII fragments found in the R.EcoRI fragment A (A, B, C, E, I) and in the R.BamHI fragment A (D, F, G, H, I, J, K) suggests that the fragment I is located in the part where the R.EcoRI fragment A and the R.BamHI fragment A overlap (Table III), this means that the series of the R.BglII fragments E-B-A is followed by the fragments C and I (Fig. 5). The R.EcoRI fragment A cleaved by R.BglII gives rise to 5 R.BglII fragments and a fragment with the molecular weight 4.5 • 106 (Table III). The R.EcoRI fragment D adjacent to the R.EcoRI fragment A (Fig. 5) is not cleaved by R.BglII. Hence, the smallest molecular weight of the uncleaved fragment should be 2.55 • 106 (1.5 • 106 + 1.05 • 106). Only the R.BglII fragment D has a higher molecular weight (3.25-106) among the R.BglII fragments which remain unassigned. Hence, the R.BglII fragment I is followed by the fragment D. Since the R.EcoRI fragment B contains fragments F, H, J and K (Table III) the R.BglII fragment D can be followed only by the fragment G which is the only one among the unmapped R.BglII fragments that is cleaved by R.EcoRI (Fig. 4.5). Since the second end fragment of the adenovirus type 6 DNA cleaved by R.BglII is the H fragment (Fig. 4.1) the remaining three fragments to be located are the fragments F, I and K which are found in the R.EcoRI fragm e n t B and, hence, in the R.HindIII fragments C2 and G (Fig. 5). The R.HindIII fragment C2 was derived from the R.BamHI fragment A, labelled with 32p by means of nick translation and cleaved by R.BglII producing the fragment J and two additional DNA fragments (Table III). This can occur only if the R.BglII fragment G is followed by the fragment J, and the R.BglII fragments J and K are separated by the fragment F. Thus, the following order is
226
B Sal
D
C
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90
100°1~
F i g . 5. T h e physical m a p o f the a d e n o v i r u s t y p e 6 D N A for the r e s t r i c t i o n e n z y m e s R . E c o R I , R . B a m H I , R . H i n d I I I and R.BglII ( p e r c e n t a g e s o f the total g e n o m e s i z e ) .
R.Sall,
suggested for the R.BglII fragments in the adenovirus type 6 DNA: E-B-A-C-ID-G-J-F-K-H (Fig. 5). Fig. 5 presents our physical maps of the adenovirus type 6 DNA for five enzymes: R. BamHI, R.EcoRI, R.SalI, R.BglII and R.HindIII. A comparison of our results with the physical maps for the DNAs of the group C adenoviruses (types 2 and 5) (Roberts, R., unpublished data) shows that the recognition sites for the above restriction enzymes on the DNAs of these adenoviruses have similar locations. However, there are certain differences, localized in definite parts (82--95%) of the genome map. This part of DNA, as one can deduce from the adenovirus functional map [30], probably codes for fiber protein, a major antigenic determinant responsible for the serotype specificity [31]. Very similar patterns of restriction sites in the left part (0--15%) of the genome of group C adenoviruses might be of interest. In adenovirus types 2 and 5 DNA
TABLE
IV
MOLECULAR WEIGHTS OF THE PRODUCTS R.BamHI FRAGMENT D BY R.HindIII
OF COMPLETE
AND PARTIAL
CLEAVAGE
C o m p l e t e cleavage f r a g m e n t s and their mol. weights ( X 1 0 -5 )
Partial cleavage fragments
Expected mol. weight ( X 1 0 -5 )
Measured mol. weight ( Y l 0 -6 )
Fragment composition
Fragment order
I.--1.24 J.--0.77 X.--O.5 Y.--0.32 M.--0.046
I II III IV V
2.01 1.85 1.29 1.09 0.55
1.96 1.8 1.3 1.03 0.56
I ,J I,M,X I,M J,Y X,M
I--J X--M--I M--I J--Y X--M
OF THE
227
this part comprises the gene(s) responsible for the transforming activity of the virus [32]. This is true also for adenovirus type 6, since R.HindIII fragment F is able to transform newborn rat kidney cells in culture (Naroditsky, B.S., unpublished results). The results obtained demonstrate a close structural similarity of adenovirus type 6, type 2 and type 5 genomes, although G + C content of adenovirus type 6 is somewhat different. This allows extrapolation of adenovirus types 2 and 5 functional mapping data to type 6. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
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